Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 149:823–832 https://doi.org/10.1007/s11240-021-02223-y ORIGINAL ARTICLE Towards a genetic model organism: an efficient method for stable genetic transformation of Eschscholzia californica (Ranunculales) Dominik Lotz1  · Jafargholi Imani2  · Katrin Ehlers1  · Annette Becker1 Received: 11 August 2021 / Accepted: 24 December 2021 / Published online: 30 May 2022 © The Author(s) 2022 Abstract California poppy (Eschscholzia californica) is a member of the Ranunculales, the sister order to all other eudicots and as such in a phylogenetically highly informative position. Ranunculales are known for their diverse floral morphologies and biosynthesis of many pharmaceutically relevant alkaloids. E. californica it is widely used as model system to study the con- servation of flower developmental control genes. However, within the Ranunculales, options for stable genetic manipulations are rare and genetic model systems are thus difficult to establish. Here, we present a method for the efficient and stable genetic transformation via Agrobacterium tumefaciens-mediated transformation, somatic embryo induction, and regeneration of E. californica. Further, we provide a rapid method for protoplast isolation and transformation. This allows the study of gene functions in a single-cell and full plant context to enable gene function analysis and modification of alkaloid biosynthesis pathways by e.g., genome editing techniques providing important resources for the genetic model organism E. californica. Key Message This work describes an efficient and reproducible in vitro method for stable genetic transformation and regeneration of California poppy (Eschscholzia californica), member of the sister lineage of eudicots. Keywords Eschscholzia californica · Ranunculales · Papaveraceae · Transformation · Protoplasts Abbreviations Introduction 2.4-D 2.4-Dichlorophenoxyacetic acid 35S 35 Svedberg units DNA fragment of the cauli- The order Ranunculales holds a key position in the phyloge- flower mosaic virus promoter region netic tree of angiosperms, because it is the sister lineage to BAP 6 -Benzylaminopurine all extant eudicots (APG IV 2016). Ranunculales emerged BIA B enzylisoquinoline alkaloids approx. 130 Million years ago (Magallón et al. 2015) and GFP Green fluorescent protein are characterized by a large diversity in life-history traits, NAA 1 -Naphthaleneacetic acid growth habit, leaf shape, flower and fruit forms, etc. and the pUBQ10 Promoter of the UBIQUITIN 10 gene of Arabi- flowers of Ranunculales in particular are extremely diverse dopsis thaliana (Damerval and Becker 2017). Besides their stunning floral VIGS V irus-induced-gene-silencing morphology, they are also extremely diverse in the produc- tion of economically valuable benzylisoquinoline alkaloids (BIAs). Around 2500 BIAS have been identified, with the major BIAs being morphine (analgesic), noscapine (anti- Communicated by M. I. Beruto. cancer drug), papaverine (spasmolytic), and sanguinarine * Annette Becker (antibacterial), and they are predominantly found in Ranun- annette.becker@bot1.bio.uni-giessen.de culales (Dastmalchi et al. 2018). 1 E. californica was developed as a model organism for the Plant Development Group, Institute of Botany, Justus- Liebig-University, Heinrich-Buff-Ring 38, 35392 Giessen, analysis of BIA metabolism and its regulation and to study Germany the evolution and functional conservation of key regulators 2 Institute of Phytopathology, Justus-Liebig-University, of flower development. Several flower developmental regula- Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany tors were analyzed already by Virus-Induced Gene Silencing Vol.:(012 3456789) 8 24 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 149:823–832 (Wege et al. 2007) providing a comprehensive view on the and efficient protocol for the isolation and transformation of regulation of floral morphogenesis in E. californica and the E. californica protoplasts and the optimization of methods conservation of the flower developmental program in dicots for plant regeneration, transformation, and generation of (Orashakova et al. 2009; Lange et al. 2013; Wreath et al. stable transgenic E. californica plants with better efficiency 2013; Zhao et al. 2018; Zhou et al. 2018). Although, VIGS than the protocol of Park and Facchini (2000b). This pro- was adopted in many Ranunculales (Damerval and Becker vides an important step towards gene function analysis in E. 2017), it is a transient method and produces sectors with dif- californica, allowing the experiments especially in the field fering gene expression, that is often not heritable (Dommes of evo-devo and BIA metabolism and its regulation. et al. 2019). Therefore a protocol to generate stable trans- genic lines of E. californica is required for many researchers working in this field. Materials and methods E. californica has provoked additional scientific interest because it is a highly invasive species. Native to mainly Cali- Plant culture, protoplast isolation fornia, it has extended its range in regions of South America, and transformation Europe, and Australia with a Mediterranean climate (Steb- bins 1965; Peña-Gómez et al. 2014; Bustamante et al. 2017). All methods described here are documented in a detailed Previous studies have shown that the fecundity of invasive step-by-step protocol on protoplast isolation, stable genetic populations of E. californica is higher than that of native transformation and in vitro regeneration, of E. californica populations and the seeds can stay viable in seed banks for and includes a trouble shooting guide (Online Resource 3). years (Peña-Gómez et al. 2014; Hickman 1993). These inva- Our step by step protocol provides the reader with a detailed sive populations are locally adapted and show a superior protocol on the sterile cultivation of E. californica. Further- colonization ability (Leger and Rice 2007). more, we provide the materials, the compositions of media Within the Ranunculales, a high-quality genome is avail- and enzyme solutions. able for opium poppy (Papaver somniferum) (Guo et al. To test vectors used for stable genetic transformation, a 2018). In addition, draft genomes are available for several protoplast system can be very useful because it provides Ranunculales members such as Eschscholzia californica and preliminary data fast. For example, different CRISPR-Cas9 Macleaya cordata of the Papaveraceae family and Aquile- constructs can be tested in the protoplast system for their gia coerulea of the Ranunculaceae family (Liu et al. 2017; genome editing efficiency. Firstly, protoplasts were isolated Hori et al. 2018; Filiault et al. 2018). These provided the from cotyledons and hypocotyl of seedlings grown in ster- base for the reconstruction of the evolutionary history of ile conditions. E. californica seeds (cv. Aurantiaca Orance enzymes involved in BIA biosynthesis (Li et al. 2020). E. King, B&T World Seeds, Aigues-Vive, France) were surface california’s major BIAs are (S)-reticuline, berberine, and sterilised by using 2.8% sodium hypochlorite solution sup- sanguinarine, however, it lacks the enzymes necessary to plemented with a drop of Tween-20 or Triton-X for 5 min synthesize noscapine and morphine (Li et al. 2020; Balažová and subsequently rinsed in sterile water (see Supplemental et al. 2020). Several regulators of the BIA biosynthesis Protocol for a detailed step-to-step protocol) and sown on (Yamada et al. 2017) or parts of the biosynthesis pathway solid full strength Gamborg B5 medium (Gamborg et al. of isoquinoline alkaloids area have been described, but 1968) including vitamins (B5, for a detailed composition of insights in BIA and isoquinoline alkaloid biosynthesis and all media, see Supplemental Material 3) supplemented with their regulation were mostly obtained by analyses utilizing 1% sucrose and 1% Gelrite (Duchefa Biochemie, Haarlem, suspension cultures (Yamada et al. 2017; Balažová et al. The Netherlands) and maintained in controlled and sterile 2020) and lack information on regulation of alkaloid bio- conditions for the entire procedure. The seeds were germi- synthesis obtained from fully grown transgenic plants of E. nated at 25 °C under long day conditions (16 h of light, light californica. A detailed protocol for suspension cultures of intensity ~ 95 µmol  m−2  s−1). The cotyledons and hypocotyls E. californica was established previously (Park und Facchini of 10-days-old seedling were used for protoplast isolation 2000a), where culture conditions were optimized by investi- based on a procedure described by Shan et al. (2014) with gating the effect of different phytohormones and component. the following modifications: about 2 g of tissue was sliced Park und Facchini (2000a, b, c) has reported a protocol to into thin strips and incubated in 0.6 M mannitol solution generate stable transgenics in this plant. for plasmolysis at RT for 10 min and transferred to 50 ml In Ranunculales species, reliable protocols for stable enzyme solution, and vacuum infiltrated (350 mmHg) for genetic transformation and subsequent regeneration of 30 min in the dark respectively. After 4 h of incubations in mature plants do not exist, with the exception of Papaver the dark with 70 rpm shaking at RT, the mixture was filtered somniferum (Yoshimatsu und Shimomura 1992; Chitty et al. through a 100 µm nylon mesh and pelleted by centrifugation 2003; Park und Facchini 2000a). Here, we present a simple at 300 g for 3 min, and resuspended in 10 ml of W5 solution 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 149:823–832 825 including 2 mM MES (pH 5.7) containing 154 mM NaCl, were compared to find the optimal levels. We analyzed the 125 mM CaCl2 and 5 mM KCl. Then protoplasts were pel- influence of different auxins of different concentrations: leted at 300 g for 3 min and resuspended in 4 ml of mannitol 2.4-D with a concentration of 0.5 mg/l up to 3 mg/l were M gCl2 (MMG, 0.4 M mannitol, 15 mM M gCl2, pH 5.7)) compared with 2 mg/l NAA in combination with 0.1 mg/l solution. Protoplast density was counted by hemocytometry. BAP. The capacity of cotyledons, hypocotyl, unripe seeds, For protoplast transformation, the working density of somatic embryos and root tissues for suitability as explant the protoplasts was adjusted to 5 × 105 protoplasts per ml were analyzed by incubation for four weeks on callus induc- with MMG solution. PEG-mediated transformation with tion medium. To get rid of the huge number of secondary the vector pGGZ003 pro35S:Omega:GFP-NLS:Dummy:T metabolites within E. californica plants, different combi- erRBCS;pMAS:BASTA:tMAS carrying 35S:GFP was per- nations of activated charcoal was tested. For maturation of formed as described in Liang et al. (2018) with the follow- somatic embryos formed, they were transferred to medium ing modifications: After the protoplast-PEG-vector mixture supplemented with 3 g/l Maltose + 2.5 mg/l ABA + 4 g/l was incubated for 20 min in the dark, the protoplasts were PEG. centrifuged at 300×g for 3 min. Following transformation, Susceptibility to antibiotics was analysed with a kill the protoplasts were incubated for about 64 h at 23 °C in the curve: E. californica calli were transferred to selection dark. Fluorescence was examined with a Leica DM5500B medium supplemented with various concentrations of microscope, a Leica DFC450 camera and the Leica L5 FL Hygromycin B (5 mg/l, 10 mg/l, 20 mg/l, 30 mg/l, 40 mg/l) Cold Light Fluorescence System Leica Filter Cube: L5. Pho- for four weeks and visually analysed. For removal of remain- tos were arranged by the Inkscape 1.0 software (Inkscape ing Agrobacteria and other potential infectious agents, callus Developers, 2020). induction medium and somatic embryo induction medium included 200 mg/l Cefotaxime. Callus regeneration and stable genetic To optimize the efficiency of plant transformation, differ- transformation ent tissues were analysed with Agrobacterium tumefaciens mediated transformation, with callus tissue being most sus- Plant regeneration and transformation was optimized by ana- ceptible. Callus tissues was immersed in A. tumefaciens lyzing several variables in the previously published protocol strain GV3101 (carrying a p35S:GFP cassette included in (Park and Facchini 2000a, b, c), for example hormone type the vector pGGZ003_p35S:GFP_HygR) suspension (with and concentration, carbohydrate composition, or the use of an OD600 of 1.0) for 20 min with mild shaking. Then, cal- activated charcoal based on literature. The optimized proto- lus tissue was transferred to callus induction medium for col commences with plant germination, where surface steri- co-cultivation with A. tumefaciens for two days in the dark, lised E. californica seeds were used. Hypocotyls of 10 days and then moved to callus induction medium with 200 mg/l old plants were used to induce callus growth on solid cal- Cefotaxime added. Subsequently after 4 weeks, the calli lus induction medium containing full strength MS + vita- were transferred to callus induction medium supplemented mins (Supplemental Material 3), 6% sucrose, 1.6% Phyto- with 200 mg/l Cefotaxime and 40 mg/l Hygromycin and agar (Duchefa Biochemie, Haarlem, Netherlands), 1 mg/l grown for four weeks on selective medium. Resistant calli 2.4-Dichlorophenoxyacetic acid (2.4-D), 5 replicates were were transferred to somatic embryo induction medium, and analysed and consisted of three seedlings each, of which the treated similarly as described above for non-transformed hypocotyl was sliced into 1–2 cm parts. The calli formed plants. Somatic embryo induction medium was supple- after 4–6 weeks were then transferred to somatic embryo mented with 200 mg/l Cefotaxime and 40 mg/l Hygromycin. induction medium including full strength B5 medium, 6% sucrose, 1.6% Phytoagar, 1 mg/l 1-Naphthaleneacetic acid Vector cloning (NAA), 0.5 mg/l 6-Benzylaminopurine (BAP), to gener- ate somatic embryos after six to eight weeks. Somatic The GFP reporter vectors pGGZ003pro35S:GFP:HygR embryos were transferred to maturation medium including and pGGZ003 pro35S:Omega:GFP-NLS:Dummy:TerR full strength B5 medium, 6% maltose, 1.6% Phytoagar, for BCS;pMAS:BASTA:tMAS were constructed using the 21 days. Subsequently, the somatic embryos were trans- Greengate system based on the vector pGGZ003 (Lam- ferred to plant regeneration medium including full strength propoulos et  al. 2013). A reaction containing 150  ng B5 medium, 6% sucrose and 1% Gelrite. Regenerated plant- of each module (pro35S:Ω:GFP:Dummy:TerRBCS; lets were transferred to soil and grown under long day con- pUBQ10: :HygromycinR: : tOCS)(pro35S:Ω:GFP- ditions (16 h of light, light intensity ~ 95 µmol m −2 s −1 at NLS:Dummy:TerRBCS; pMAS:BASTA:tMAS) and 100 ng 22 °C). of pGGZ003 were set up according to Lampropoulos et al. During the optimization process of the plant regenera- (2013). Chemocompetent E. coli DH5α cells were trans- tion protocol, different concentrations of media components formed with 4 µl reaction mix to amplify the construct. The 1 3 8 26 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 149:823–832 correct assembly was monitored by sequencing and the 13.5 and 24.8% (Fig. 1C). The transformation efficiency vector was isolated and introduced in A. tumefaciens strain is dependent on the incubation time of the enzyme solu- GV3101 by electroporation following standard protocols. tion, such that an incubation time of 1.5 h yielded 11.8% transformed protoplasts and 4 h 24.8% transformed proto- Western blot analysis plasts (Fig. 1D). Incompletely digested cell walls may thus inhibit the PEG-mediated transformation. Nuclear localized Tissue from putatively transgenic E. californica plants GFP signal was detected using fluorescent microscopy 64 h was collected from different developmental stages (callus, post transformation (Fig. E–G). Protoplasts were visually somatic embryos, young regenerated plants) during plant unchanged (Fig. 1E) when compared to freshly isolated pro- regeneration. Material was frozen in liquid nitrogen and toplasts (Fig. 1A) suggesting that the transformation and ground using a tissue-lyser. The sample was then mixed subsequent incubation time had no obvious negative effect with pre-heated 4 × Laemmli buffer (100 µl/200 mg sam- on protoplast viability. The protoplasts showed a very low ple containing 30 mg/ml DDT). After 5 min at 100 °C, the level of auto fluorescence (Fig. 1F). Online Resource 1, samples were centrifuged at 14,000 rpm for one minute Fig. 1 provides a flowchart summarizing the experimental and 30 µl of the supernatant was loaded on a 12% SDS gel. steps and expected time line for protoplast transformation PageRuler Prestained Protein Ladder (Thermo Fischer, Lan- and all other approaches described here. genselbold, Germany) was used as a marker. After 30 min at 80 V followed by 2 h at 100 V) the gel was transferred Plant regeneration from explants to a PVDF membrane. The samples were blocked with 1% BSA in TBS buffer + Tween for 2 h and incubated with an Establishing E. californica as a genetic model plant requires Anti-Green Fluorescent Protein – Horseradish Peroxidase the possibility for stable transformation and thus we devel- (Miltenyi Biotec, Bergisch Gladbach, Germany) (GFP-HRP) oped a reliable, tissue-culture based method for plant regen- antibody 1:8000 in 2.5% BSA overnight at 4 °C with shak- eration as a first step towards a transformation protocol. ing. After the incubation, the gel was washed three times for We first analyzed the type of explant, and type and con- 10 min each in TBS-T (0.1% TWEEN20) solution. To detect centration of auxin best suitable for callus regeneration of E. the proteins, 500 µl of Amersham ECL Western Blotting califonica (Table 1). Using 2.4-D as auxin source resulted Detection Reagent (1:1 solution A and solution B, Amer- in callus regeneration in all the tested concentrations, with sham, Freiburg, Germany) was used. Images were created hypocotyl being more amenable to callus regeneration. A using a ChemiDOC MP Imaging System (Biorad, Neuberg, concentration of 1 mg/l 2.4-D was sufficient for regenera- Germany). tion of callus tissue from every explant. After four weeks, almost all hypocotyl explants regenerated callus (97.8%) that were subsequently induced to develop somatic embryos. Results and discussion The details of the following experiments can be found in Online Resource 2, Table 1 to 6). After seven weeks, 44% Protoplast isolation and transformation of the calli produced somatic embryos and after this time, only marginally more calli generated somatic embryos. The Many experimental approaches, such as biochemical, pro- somatic embryos were removed from the calli and matured tein–protein or protein-DNA interaction analyses do not for three weeks on a specific maturation medium includ- require fully-grown plants and protoplasts are suitable exper- ing maltose instead of sucrose and subsequently, they were imental systems. We thus developed a protocol for rapid kept in the light until they turned green and commenced protoplast isolation and transformation (Online Resource 3, organogenesis. From 58% of the somatic embryos, plantlets step-by-step protocol). Protoplasts were isolated from the could be regenerated, and 60% of these plantlets developed leaf and hypocotyl tissue of 10 days old in vitro grown plants into fully grown plants in soil. The regenerated plantlets using macerozyme and cellulase R-10 to digest cell walls. were transferred to soil after 4 to 6 weeks. The surviving The protoplast isolation yielded between 0.45 and 0.85 × 1 06 plants formed morphologies similar to plants grown from protoplasts/g plant tissue. These protoplasts were round, seeds and were fully fertile. Generally, the regeneration of E. only occasionally was the cell wall still attached, and they californica plants was carried out in at least five repititions. included many chloroplasts (Fig. 1A, B). Taken together, we established an efficient E. californica Next, we transformed the freshly isolated protoplasts in vitro plant regeneration protocol, from hypocotyl explants with a nuclear localized, ubiquitously expressed GFP to mature plants via callus induction and somatic embryo- protein as fluorescent marker driven by UBQ10 promoter genesis. Figure 2 shows an overview of the time scale and (pUBQ10:GFP:NLS). Employing a PEG-mediated transfor- morphology of E. californica regeneration intermediates. mation protocol, the transformation efficiency was between 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 149:823–832 827 Fig. 1 Protoplast isolation and transformation. Morphol- ogy of freshly isolated E. californica leaf and hypocotyl protoplasts (A, B). C Proto- plast isolation efficiency in relation to the percentage of pUBQ10:GFP:NLS carry- ing, fluorescing protoplasts. D Incubation time in cell-wall dissolving enzyme solution affects transformation effi- ciency. Bars represent standard deviation, based on at least five independent repetitions. E Protoplasts transformed with pUBQ10:GFP:NLS in brightfield, fluorescence (F), and overlay (G). Size bars are 100 µm Optimization of the plant regeneration protocol regeneration rate than cotelydons and hypocotyl, independ- end of the auxin source (Table 1).We also analyzed if reduc- To obtain an efficient and reproducible protocol for E. cali- ing cell culture inhibitory secondary metabolites by adding fornica transformation, several modifications to the transfor- 0.5 g/l activated charcoal to the medium improves callus mation protocol reported earlier (Park and Facchini 2000b) formation (Möller et al. 2006; Thomas 2008). However, the were tested. For example, the addition of 2.0 mg/l NAA and callus regeneration from hypocotyl dropped from 100 to 33% 0.1 mg/l BAP to the callus induction medium was replaced when charcoal was added (Table 1), suggesting that charcoal by adding only 1 mg/l 2,4 D, which raised the number of may block phytohormone activity or availability. Addition- explants producing calli to nearly 100% (Table 1). Further, ally, we analysed if somatic embryos can dedifferentiate to we analyzed if cotyledons or hypocotyl produced callus callus by adding 1 mg/l 2.4-D or 2 mg/l NAA, 0.1 mg/l BAP material more reliably and found that with the optimal con- and activated charcoal. 2.4-D leads to new callus regenera- centration of 1 mg/l 2,4-D, all hypocotyl explants produced tion in more than 90%. Interestingly, the combination of callus while only max. 80% of the cotyledon explants pro- NAA, BAP and activated charcoal leads to the regeneration duced calli (with an optimal 2,4-D concentration of 1,5 mg/l, of new somatic embryos within six weeks in nearly 100% of Table 1). Next we analysed if unripe seeds (21 days after all samples (Table 1). Finally, a somatic embryo maturation polination) have the potential to regenerate callus. We com- phase was introduced to the protocol to improve greening pared 1 mg/l 2.4-D with 2 mg/l NAA and 0.1 mg/l BAP and proper morphogenesis of the embryos. We analyzed sev- and our results show that unripe seeds have a lower callus eral combinations of maltose, PEG and abscisic acid (ABA), 1 3 828 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 149:823–832 Table 1 Callus regeneration rate dependent on different E. californica organ explants and varying concentrations of 2,4-D, NAA, BAP and 0.5 g/l activated charcoal Cotyledons as explants for callus production Cotyledons as explants for callus production mg/l Total number Callus Dead tissue Callus regen- mg/l 2,4-D + acti- Total number Callus Dead tissue Callus 2,4-D eration (%) vated charcoal regeneration (%) 0.5 8 4 4 50.00 0.5 8 1 7 12.50 1 13 3 10 23.08 1 8 2 6 25.00 1.5 5 4 1 80.00 1.5 5 0 5 0.00 2 5 1 4 20.00 2 4 0 4 0.00 3 6 4 2 66.67 3 4 1 3 25.00 Hypocotyls as explants for callus production Hypocotyls as explants for callus production mg/l Total number Callus Dead tissue Callus regen- mg/l 2,4-D + acti- Total number Callus Dead tissue Callus 2,4-D eration (%) vated charcoal regeneration (%) 0.5 7 5 2 71.43 0.5 9 0 9 0.00 1 4 4 0 100.00 1 3 1 2 33.33 1.5 5 3 2 60.00 1.5 5 1 4 20.00 2 4 4 0 100.00 2 2 0 2 0.00 3 7 7 0 100.00 3 4 0 4 0.00 Unripe seeds as explants for callus production Somatic embryos as source for callus production Total number Callus Dead tissue Callus Total number Callus New SEs Root Callus regenera- hair regeneration tion (%) (%) 1 mg/l 310 70 240 22.58 1 mg/l 26 24 7 – 92.31 2,4-D 2,4-D 2 mg/l 204 81 123 39.71 2 mg/l 24 7 23 9 29.17 NAA + 0.1 mg/l NAA + 0.1 mg/l BAP BAP + activated charcoal Fig. 2 Time scale of plant regeneration from E. californica hypocotyl were transferred for three weeks to maturation medium. After matura- explants. Hypocotyl of 10 days old E. californica plants was used to tion, all embryos were incubated in long day conditions (16  h light induce callus growth on callus medium. After 6–8 weeks in the dark 8 h dark) on plant regeneration medium without hormones. Regener- at 25 °C, regenerated callus cells were transferred to somatic embryo ated plants were transferred to soil, covered with a cap for two weeks, induction medium. For embryo maturation, regenerated embryos to maintain humidity and found that exchanging sucrose as carbon source with of the calli (Online Resource 2, Table 3). In addition, the 3% maltose alone performed best, with around 84% of the regenerated plants grew better in plain standard soil; around embryos started photosynthesis in contrast to a combination 80% survived the transfer from in vitro culture to soil when of 3% maltose with 2.5 mg/l ABA which killed around 97% 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 149:823–832 829 compared to vermiculite as substrate, which only around the flowers, which show folded mature petals in the trans- 54% of the plants survived (Online Resource 2, Table 6). genics (Online Resource 1, Fig. 3). Agrobacterium‑mediated transformation of E. Other E. californica transformation methods californica There are other means to generate transgenic E. califor- Based on the tissue culture and regeneration method nica tissue, for example by using biolistic bombardment of described earlier, we developed an efficient transforma- callus tissue (Popelka et al. 2003; Viehweger et al. 2006; tion protocol using E. californica hypocotyl as explant and Angelova et al. 2010), but this requires an expensive biolis- employing Agrobacterium tumefaciens based transforma- tic particle delivery system. Using the regeneration proto- tion to generate transgenic lines carrying the reporter con- col reported here, this method can be extended to generate struct p35S::GFP using hygromycin as selectable marker. transgenic lines. Park and Facchini (2000b) has reported A killing-curve experiment showed that a concentration of another protocol, but with different media/phytohormone 40 mg/l hygromycin is sufficient to select transgenic plants compositions and less efficient in terms of callus induc- (Online Resource 1, Fig. 2; Online Resource 2, Table 7). tion frequency (80%) than the method standardized in this The transgenic calli produced somatic embryos, which were study (98%). Also the somatic embryo formation from cal- then regenerated into fully grown, fertile plants employing lus was improved (45%) than in the previous study (30%). the plant regeneration protocol. Figure 3A–P shows mock- Further, the somatic embryo induction medium we used treated and 35S:GFP treated calli and somatic embryos in could induced continuous production of somatic embryos bright field and fluorescence microscopy. Only a small frac- from transgenic calli, such that up to 37 transgenic lines tion of callus cells is transformed and has already started to could be subcultured from a single callus (Online Resource divide (Fig. 3A–H). Those will later provide the transgenic 2, Table 8). Unfortunately, we cannot compare our work with calli shown in Fig. 3L and P, while the untransformed cells how the method by Park and Facchini (2000b) performs in of the calli discontinue division and die eventually. Healthy other labs, as no further work based on this protocol was calli under selection are then removed and incubated on published. somatic embryo induction medium. Wild type calli regen- erating somatic embryos are shown in Fig. 3I, J, M, N). Fig- ure 3K, L, O, and P show two fully fluorescent calli that are Conclusions derived from transformation events. Those generate geneti- cally identical somatic embryos that are also completely The possibility to create stable transgenics is the prerequi- fluorescent, showing that the 35S promoter is active in cal- site to establish E. californica as a genetic model system lus tissue and somatic embryos. The presence of transgenic for the Ranunculales, the sister order to the core eudicots. GFP protein in the transformed tissues was corroborated by Other practical aspects such as a short generation cycle of Western blot analysis (Fig. 3Q). In total, 23 independently only two months from seed to flower, undemanding grow- transformed transgenic calli were derived that, when cul- ing conditions, and a comparatively small genome six times tured on somatic embryo induction medium, all continuously that of Arabidopsis thaliana add to the value of the model produced genetically identical somatic embryos. A strong species E. californica (Becker et al. 2005; Wege et al. 2007; GFP signal was detected in 65% of the regenerated somatic Yamada et al. 2021). Our work provides efficient means for embryos (Fig. 3 and Online Resource 2, Table 8). gene function analysis in E. californica using stable trans- Unexpectedly, the mock-treated calli and somatic genic lines to generate reproducible results. Approaches like embryos also show a faint fluorescence (Fig. 3B, F, J, N), CRISPR-Cas9 mediated genome modifications, reporter but this is much lower in intensity than the GFP fluorescence lines for phytohormones detection, or stable and localized in Fig. 3C, D, G, H, L, and P, and suggestive of fluorescing modifications of alkaloid biosynthesis (Alagoz et al. 2016; secondary metabolites. Interestingly, it was shown previ- Isoda et al. 2021; Hayashi et al. 2020) could be studied with ously, that E. californica suspension cultures produce benzo- the help of the protocol developed in this study. Previous phenanthridine alkaloids with an excitation of 270–550 nm work (Park and Facchini 2000b) provided a protocol with and emissions at 310–590 nm (Hisiger and Jolicoeur 2005). lower efficiency regarding the regeneration of E. californica Those lie within the range of the GFP excitation and emis- plants and did not consider protoplast isolation and trans- sion spectrum (max. 488 nm and 507 nm, respectively) and formation. Researchers interested in alkaloid biosynthesis could explain the low intensity fluorescence observed in and regulation may work with E. californica without spe- wild type calli and somatic embryos. p35S:GFP transformed cific permits as they are required for P. somniferum. For plants show a morphology similar to that of wild type plants example, E. californica could be employed as a promising that underwent tissue culture based regeneration, except for model species to study the mutual influence of development 1 3 830 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 149:823–832 Fig. 3 Documentation of transformed E. californica plants during fluorescent light. Q Western blot analysis of transgenic E. californica regeneration. A–H shows examples of growing calli. A and E, mock- tissue (1) wild type callus, (2) callus carrying 35S:GFP, (3) somatic treated calli in bright field. B and F, same calli under fluorescent embryos carrying 35S:GFP, (4) mixed sample from young regener- light. C, D, G and H, calli transformed with 35S:GFP under fluores- ated plants and somatic embryos, (5) GFP antibody positive control: cent light. I and M, mock-treated somatic embryos in bright field, J tobacco leaves Agrobacterium-infiltrated with HA-GFP tag that were and N same embryos in fluorescent light. K and O calli and somatic previously analyzed to express GFP. Green arrows point to the GFP- embryos carrying 35S:GFP in bright field, L and P same tissues in HA bands and alkaloid biosynthesis (Yamada et al. 2020) or to analyse Supplementary Information The online version contains supplemen- flower evolution on the molecular level (Zhao et al. 2018). tary material available at https://d oi.o rg/1 0.1 007/s 11240-0 21-0 2223-y. Taken together, our work provides an important step towards the establishment of E. californica as a genetic model sys- Acknowledgements We thank Dietmar Haffer for his expertise and help in transferring the in vitro grown plants to soil, Thomas Groß tem to study a large suite of diverse research questions. 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2022) 149:823–832 831 for constructing the vector carrying the 35S:GFP cassette and Mar- supplementation. Mol (basel, Switzerland) 25(6):1261. https:// tina Claar for support with the Western blot. The student Verena Gisa doi.o rg/1 0. 3390/m olecu les2 50612 61 helped with tissue culture. Becker A, Gleissberg S, Smyth DR (2005) Floral and vegetative mor- phogenesis in California poppy (Eschscholzia californica Cham.). Author contributions DL, KE and JI carried out experiments and Int J Plant Sci 166(4):537–555. https:// doi.o rg/ 10. 1086/ 429866 analysed the data; JI and KE gave input in experimental design and Bustamante RO, Durán AP, Peña-Gómez FT, Véliz D (2017) Genetic helped with microscopy; AB supervised experiments and wrote the and phenotypic variation, dispersal limitation and reproductive manuscript. All authors read and approved the final version of the success in the invasive herb Eschscholzia californica along an ele- manuscript. vation gradient in central Chile. Plant Ecol Divers 10(5–6):419– 429. https:// doi. org/1 0. 1080/ 17550 874.2 018.1 42550 4 Chitty JA, Allen RS, Fist AJ, Larkin PJ (2003) Genetic transforma- Funding Open Access funding enabled and organized by Projekt DEAL. This work was funded by the Justus-Liebig-University Gießen, tion in commercial Tasmanian cultivars of opium poppy, Papaver Germany. somniferum, and movement of transgenicpollen in the field. Funct Plant Biol 30(10):1045–1058. https://d oi. org/ 10. 1071/ Fp031 26 Damerval C, Becker A (2017) Genetics of flower development in Data availability Vectors and seeds of transgenic lines may be obtained ranunculales: a new, basal eudicot model order for studying flower from the corresponding author. evolution. New Phytol 216(2):361–366. https:// doi. org/ 10. 1111/ Nph.1 4401 Code availability n.a. Dastmalchi M, Park MR, Morris JS, Facchini P (2018) Family por- traits: the enzymes behind benzylisoquinoline alkaloid diver- Consent for publication n.a. sity. Phytochem Rev 17(2):249–277. https:// doi. org/ 10. 1007/ S11101- 017-9 519-Z Dommes AB, Gross T, Herbert DB, Kivivirta KI, Becker A (2019) Declarations Virus-induced gene silencing: empowering genetics in non-model organisms. J Exp Bot 70(3):757–770. https://d oi.o rg/1 0.1 093/J xb/ Ery411 Conflict of interest There are no conflicts of interest or competing in- terests. 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