Gp. lemaneiformis tertiary branch tips were cut out (about 1-cm long) and grown under the same temperature and light conditions as described above. They were grown in 12-well plastic plates with lid (Corning Inc., USA) in 5 ml of seawater with Pro medium per well. For preliminary screening, the wells contained 1; 0.5 or 0.1 mg.ml^-1 of Cm, Hyg, PMM or Zeo (Solarbio Science & Technology Co., Ltd, Beijing, China). For the determination of working conditions, they contained 1; 0.5 or 0.25 mg.ml^-1 of Hyg or PMM. Six branch tips from different individuals per genotype were put in presence of each antibiotic (three tips per well) during 15 days. The control wells contained seawater with Pro medium without any antibiotic. All the different media were adjusted to Ph 8.8 ± 0.1 using NaOH. The medium was changed every 5 days.

The viability of the branch tips was determined by their color. The color of Gp. lemaneiformis ZC and WT individuals is dark red when they are alive, but becomes white or yellow when they die as the concentration of photosynthetic pigments decreases. The tips were scored as dead when they became completely white or yellow.

The inhibitory concentration 50 (IC₅₀), used to determine the concentrations of Hyg required to inhibit the growth of Gp. lemaneiformis, was calculated using the online tool ICEstimator (biostat.fr) (Kaddouri et al., 2006; Le Nagard et al., 2020). The 95% interval was obtained from the same online tool. The method used for estimating IC₅₀ applies the following model:

where RE is the relative effect of the antibiotic, C is the antibiotic concentration, and γ is the sigmoidicity factor.

The maps of the plasmids purchased from external sources are presented on Supplementary Figure S7 , and those of the plasmid cloned during this project are presented on Supplementary Figure S8 (designed with Snapgene Viewer 5.1, GSL Biotech LLC, USA).

The pGFPGUSPlus plasmid (Vickers et al., 2007) was ordered from Addgene (Watertown, MA, USA). Its sequence was verified by Sanger sequencing using the M13F20, GFP-F/R, and Hpt-F/R primers (see Supplementary Table S1 ) and by enzymatic digestion using the EcoRI and HindIII restriction enzymes (Thermo Fisher Scientific, USA) (see Supplementary Table S4 , Supplementary Figure S9 ).

The JRH0647 plasmid was kindly provided by Pr Fanna Kong (Ocean University of China). Its quality was verified by simple gel electrophoresis ( Supplementary Figure S10 ) and by Sanger sequencing using the JRH0647-F/R primers.

The mTagBFP2-N and pA7-GFP expression vectors were purchased from Miaoling Biology (Hangzhou, China). They were verified by enzymatic digestion ( Supplementary Figure S11 ) and Sanger sequencing using the GFP-F/R and BFP-F/R primers.

The R0097-07N plasmid containing the EGFP sequence under the control of the GlAct1 promoter was cloned by Synbio Technologies (Suzhou Hongxun Biotechnologies CO., LTD) using the pGFPGUSPlus vector backbone, synthesizing the GlAct1 promoter sequence and inserting it in front of EGFP instead of the CaMV35S promoter. It was verified by enzymatic digestion ( Supplementary Figure S11 ) and Sanger sequencing using the GFP-F/R and Hpt-F/R primers.

The GBFP-pA7 plasmid containing the mTagBFP2 reporter gene under the control of the GlAct1 promoter and the PBFP-pA7 plasmid containing the mTagBFP2 reporter gene under the control of the PyAct1 promoter were constructed by first obtaining intermediate GGFP-pA7 and PGFP-pA7 vectors. Briefly, these intermediate vectors were obtained by amplifying the GlAct1 and PyAct1 promoters from the R0097-07N plasmid and the P.yezoensis genome, respectively, using primers introducing HindIII and XhoI restriction sites upstream and downstream the promoter fragments (GlAct1-F/R and PyAct1-F/R primers, see Supplementary Table S1 , Supplementary Figure S12A ). The resulting PCR products and the pA7-GFP vector were then simultaneously digested with HindII and XhoI ( Supplementary Figure S12B ) and ligated with the T4 DNA ligase to obtain the GGFP-pA7 and PGFP-pA7 intermediate vectors containing the EGFP gene under the control of either the GlAct1 or the PyAct1 promoters instead of the CaMV35S promoter. The two vectors were then verified by colony PCR ( Supplementary Figure S12C ) and Sanger sequencing.

To obtain the final GBFP-pA7 and PBFP-pA7 plasmids, the mTagBFP2 reporter gene sequence was amplified from the mTagBFP2-N expression vector template, using primers introducing XhoI and BamHI restriction sites upstream and downstream the target fragment (BFP-F/R primers, see Supplementary Table S1 , Supplementary Figure S12A ). The mTagBFP2 PCR fragment and the GGFP-PA7 and PGFP-PA7 intermediate vectors were then double digested with XhoI and BamHI ( Supplementary Figures S12B , S13A ), and the digested mTagBFP2 fragment was ligated to respectively the GGFP-PA7 and PGFP-PA7 digested vectors, following the same procedure as above. The final GBFP-pA7 and PBFP-pA7 plasmids expressing the mTagBFP2 reporter gene under the control of either the GlAct1 or the PyAct1 promoters were thereby obtained before being verified by colony PCR ( Supplementary Figure S13B , S13C ) and Sanger sequencing.

All DNA concentrations were measured using a nanodrop (Implen, Munich, Germany). DH5α E. coli transformed with the different plasmids were grown at 37°C in LB medium containing the appropriate antibiotics (pGFPGUSPlus, R0097-07N, pmTagBFP2-N: 50 μg/ml Kanamycin [Kan], JRH0647: 25 μg/ml Chloramphenicol [Cm], pA7-GFP, GGFP-pA7, PGFP-pA7, GBFP-pA7, PBFP-pA7: 50 μg/ml Ampicillin [Amp]). Fifty percent glyerol stocks were kept. The plasmids were extracted using the TIANprep Mini Plasmid Kit (Tiangen Biotech, Beijing, China).

The plasmid sequences are presented in the Supplementary Data ( Supplementary Sequences S1–S5 ).

As no Actin promoter sequence was known in Gracilariopsis lemaneiformis, a BLAST search on the Gp. lemaneiformis genomic database (Zhou et al., 2013) was carried out using the Gracilaria textorii beta-actin mRNA sequence (GenBank: EU095962.1). The SRR1011281.38131 sequence was identified on the Gp. lemaneiformis chromosome 3, with a 63% query cover and 72% identity ( Supplementary Sequence S8 ). After having translated the putative Actin sequence using the ExPAsy Translate tool (https://web.expasy.org/translate/) ( Supplementary Sequence 9 ), a BLAST query on the NCBI protein database yielded the following sequences (the top three sequences are described): Actin (Chondrus crispus), Ref. Seq. XP_005717262.1, 100% query cover, 94% identity; Actin (Gracilariopsis chorda) Ref. Seq. PXF4927.1, 100% query cover, 90% identity; Actin 1 (Nemalionopsis shawii) Ref. Seq. CAI56221.1, 96% query cover, 95% identity.

Another BLAST search on Gp. lemaneiformis genome database was carried out using the P. yezoensis Act1 mRNA sequence (GenBank: AB455256.1). The same sequence as obtained with the Gracilaria textorii beta-actin mRNA sequence ( Supplementary Sequence S8 ) was found, with a 93% query cover and 76.16% identity. Another sequence, SRR1011281.3103 ( Supplementary Sequence S10 ) was identified with a 98% query cover and 73.13% identity and positioned on chromosome 22. After having translated the putative Actin sequence using the ExPAsy Translate tool ( Supplementary Sequence S11 ), a BLAST query on the NCBI protein database yielded the following sequences (the top three sequences are described): Actin (Gracilariopsis chorda) Ref. Seq. PXF4927.1, 100% query cover, 100% identity; Actin (Chondrus crispus), Ref. Seq. XP_005717262.1, 99% query cover, 92.45% identity; Hypothetical protein BU14_0031s0095 (Porphyra umbilicalis), 100% query cover, 86.10% identity.

To select the most suitable Gp. lemaneiformis Actin promoter, the sequences upstream the Actin coding sequences identified were analyzed with the TSSP Prediction of plant promoters’ tool (http://linux1.softberry.com/berry.phtml?topic=tssp&group=programs&subgroup=promoter), but no significant promoter sequence was identified. Upstream (200 bp) the coding sequences were therefore selected and compared with the CGI gene analysis tool (http://218.4.117.30:3000/cgi-bin/gene_analysis.cgi). The SRR1011281.38131 promoter sequence had a total GC content of 53.30%, seven direct and seven reverse repeats, whereas the SRR1011281.3103 promoter sequence had a total GC content of 58.13%, six direct and 13 reverse repeats. The SRR1011281.38131 promoter sequence was thus less complex and was chosen as it was assumed to be easier to clone. The 200 bp upstream the coding sequence was named Gp. lemaneiformis Actin 1 (GlAct1) promoter and was cloned upstream the EGFP sequence in the pGFPGUSPlus vector by Synbio Technologies.

Thirty milligram of 0.6-μm diameter gold particles was suspended in 1 ml of 70% ethanol and vortexed for 5 min. After letting them stand in the solution for 15 min at room temperature, they were centrifuged at 1500 rpm for 5 min. The supernatant was then discarded and the particles were washed three times by adding 1 ml of sterile water, vortexing for 1 min, and centrifuging at 1,500 rpm for 5 min. The supernatant was then discarded and the particles were resuspended in 500 μl of 50% glycerol. They were stored at −20°C. The plasmids to be bombarded were extracted with the TIANprep Mini Plasmid Kit (Tiangen Biotech, Beijing, China) and concentrated to 1 μg/μl using a vacuum concentrator centrifuge (MSLNC01). To coat the plasmids on the gold particles, 17 μl of the gold particle suspension were added to 6 μl of plasmid, 17 μl of 2.5 M CaCl₂, and 6.8 μl of 0.1 M spermidine, before being sequentially vortexed 1 min and kept on ice 1 min 10 times in a row. The suspension was then kept on ice for 30 min before being centrifuged at 10,000 rpm for 10 s. The supernatant was removed, and the coated gold paticles were sequencially washed with 140 μl of 70% ethanol, 140 μl of absolute ethanol, and resuspended in 60 μl of absolute ethanol. The same protocol was repeated for the negative control without plasmid. One centimeter–long Gp. lemaneiformis tertiary branch tips were placed in the center of polystyrene 10-cm-diameter petri dishes. The tips were organized on a single layer, forming a circle of 2-cm diameter. DNA transformation was carried out by particle bombardment using the Biolistic PDS-1000/He particle acceleration device (Bio-Rad, USA), according to the manufacturer’s instructions. Twenty-four microliter of coated gold particles was used for each shot at a 6-cm target distance at 28 in. Hg of vacuum pressure and 1,100 psi of helium pressure, through a rupturable membrane of 1,350 psi. The transformed tips were kept in the dark for 1h without water, and sterile seawater complemented with Pro medium was then added. The tips were kept in the dark for 24h after bombardment, before being grown under normal conditions (see above). Pro medium was changed every day.

For the DNA transformation, 150 tips were placed in each dish; three dishes were transformed with the pGFPGUSPlus plasmid and three with the control uncoated gold particles. Fifty tips of each were randomly selected 2–8 days after bombardment to extract DNA.

One hundred fifty tips bombarded with the JRH0647 plasmid or the control uncoated gold particles were randomly picked 36h and 48h after bombardment and kept at −80°C during a few weeks before extracting RNA. Similarly, 60 tips bombarded with the pGFPGUSPlus plasmid or with the control uncoated gold particles were collected every day from the 2^nd to 8^th day after bombardment. Fifty tips bombarded with either the GBFP-PA7 plasmid, the PBFP-PA7 plasmid, or the control uncoated gold particles were collected every day 2, 4, and 6 days after bombardment. Fifty tips of each group were kept at −80°C during a few weeks before carrying out RNA extraction, whereas the remaining tips were used for GUS staining or Bfp fluorescence observation on the day of collection.

Two hundred tips were placed in petri dishes and transformed with the pGFPGUSPlus plasmid and 200 with the control uncoated gold particles. One hour after bombardment, the algal tips were grown in sterile seawater complemented with Pro medium. The bombarded dishes were grown in presence of 0.5 mg.ml^-1 Hyg during 20 days. The tips were kept in the dark for 24h after bombardment.

The same experiment was then repeated with 1 mg.ml^-1 Hyg. Five hundred tips transformed with the pGFPGUSPlus plasmid (125 tips in each of the four dishes) and 500 with the control uncoated gold particles. One hour after bombardment, the algal tips were grown in sterile seawater complemented with Pro medium. The tips were kept in the dark for 24h, and 1 mg.ml^-1 Hyg Hyg was added 24h after bombardment. The tips were treated with Hyg during 10 days and transferred to normal seawater complemented with Pro until they all died after 15 days. The dead tips were scored every day.

One hundred milligram of Gp lemaneiformis branch tips were grinded using the Jingxin Tissuelyser-24 (Shanghai Jingxin Industrial Development, Shanghai, China), after having added liquid Nitrogen, following the manufacturer’s instructions. RNA was then extracted using the E.Z.N.A Total RNA kit (Omega Bio-Tek, USA) following the manufacturer’s instructions. DNA was extracted using the TIANGEN Plant Genomic DNA kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions, and treated with DNAse ( Supplementary Table S4 ).

Before extracting DNA after microparticle bombardment to check the presence of the plasmids, it was necessary to ensure that there was no plasmid in vitro outside the tips, to avoid false positives. The tips were then treated with DNAse I (Thermo Fisher Scientific, USA) directly in Eppendorff tubes ( Supplementary Table S4 ).

cDNA was generated from total RNA using the Evo M-MLV reverse-transciption kit (Accurate Biotechnology, Hunan, China) following the manufacturer’s instructions. RNA (1.5 μg) was used in each 20-μl reaction.

Primers described here are presented on Supplementary Table S1 . PCR reaction components are presented on Supplementary Table S2 . Thermal cycling conditions are presented on Supplementary Table S3 . To check the presence of the pGFPGUSPlus plasmid in the tip cells after microparticle bombardment, PCR was carried out using the HygR-F/R and GFP-F/R primers on total DNA extracted from bombarded tips.

The expression of the Hpt gene from the pGFPGUSPlus plasmid, and the mTagBFP2 gene from the GBFP-pA7 and PBFP-pA7 plasmids in the bombarded tips, was checked by RT-PCR on their cDNA. The cDNA was amplified using the HygR-F/R, GFP-F/R and BFP-F/R primers. The Gapdh gene was used as standard; it was amplified using the primers GAPDH-F/R. The gRNA expression from the JRH0647 plasmid was checked on the cDNA extracted from bombarded tips using the JRH0647-F/R primers.

Ten algal tips were randomly collected 5 days after microparticle bombardment with the pGFPGUSPlus plasmid or the control uncoated particles, and 2-mm thick-cross sections were performed on the tips before proceeding to the GUS staining reaction. Ten 1 cm² onion inner epidermis sections were also bombarded and used as a positive control for GUS expression. The staining solution was obtained with a GUS staining kit (Solarbio G3060, Beijing, China) following the manufacturer’s instructions. Twenty microliter of X-Gluc solution was mixed with 1 ml of GUS buffer. It was protected from light and stored at 4°C for 1 day before staining. Three hundred thirty microliter of GUS staining solution was used to completely submerge the tips and the onion epidermis in 1.5-ml EP tubes. The tubes were wrapped in aluminum foil to protect the samples from light and were incubated at 37°C for 20h. The material was then incubated for 1.5h in 70% ethanol for decolorizing the tissues. The tissues were then observed under an Olympus BX53 microscope using 40× magnification, and pictures were taken with a Mshot MD30 camera; the sites of GUS activity appeared blue.

Fifty tips bombarded with the GBFP-PA7 and PBFP-PA7 plasmids and 50 unbombarded tips (negative controls) were collected on the 2^nd, 4^th, and 6^th days after transformation. They were observed under a Nikon 80i fluorescence microscope under blue fluorescence excitation filter using DAPI mode (380–420 nm) to observe the expression of the mTagBFP2 gene in the cells.

To develop a selection system for genetically transformed cells, the sensitivity of Gp lemaneiformisto various antibiotics was tested.

Branch tips were cut out and grown in the presence of 0.1, 0.5, or 1 mg.ml^-1 of Cm, Hyg, PMM, or Zeo during 15 days ( Figure 1 ). Six branch tips from different individuals per genotype were put in the presence of each antibiotic (three tips per well). The tips were considered dead when they became completely yellow and were counted every day to determine the mortality rate.

Cm did not have any significant effect on Gp. lemaneiformis tips, as only diploid tips started to die after 11 days at the lowest concentration ( Figures 1A, B , Supplementary Table S5 , Supplementary Figure S1 ). On the other hand, the diploid and haploid tips treated with 0.5 mg.ml^-1 of Hyg started to die after 9 and 7 days, respectively ( Figures 1C, D ). They started to die after 7 days when treated with 1 mg.ml^-1 Hyg, and all the tips treated with 0.5 or 1 mg.ml^-1 were dead after fifteen days ( Supplementary Table S5 , Supplementary Figure S3 ). The mortality of the tips treated with 0.1 mg.ml^-1 Hyg did not increase significantly, as only two haploid tips were dead after fifteen days ( Figures 1C, D , Supplementary Table S5 , Supplementary Figure S3 ). PMM also seemed to affect the tips, as the diploid individuals treated with 0.5 and 1 mg.ml^-1 PMM started to die after 11 and 7 days, respectively, and after 9 and 7 days for haploid tips ( Figures 1E, F ). There was no significant increase in mortality at 0.1 mg.ml^-1. All the diploid tips were dead after 15 days at 0.5 and 1 mg.ml^-1, whereas only one haploid tip survived ( Supplementary Table S5 , Supplementary Figure S4 ). Finally, Zeo did not have any significant effect on the tips’ survival, as only two control diploid tips died ( Figures 1H , Table 1 , Supplementary Figure S2 ).

This test suggests that Hyg and PMM negatively affect the survival of Gp. lemaneiformis branch tips at concentrations of 0.5 and 1 mg.ml^-1. They were therefore selected for further screening.

After Hyg and PMM were selected for screening, a new set of treatment was performed to confirm the results and choose between the two antibiotics. As a concentration of 0.1 mg.ml^-1 did not show any significant effect on the tips’ survival, the treatment was only repeated with concentrations of 0.5 and 1 mg.ml^-1. Hyg was confirmed to negatively affect Gp. lemaneiformis branch tip survival as all the treated tips were dead after fifteen days of treatments, whereas only one non-treated haploid tip had died ( Table 1 , Supplementary Figure S5 ). PMM was also found to affect tip survival at 1 mg.ml^-1 but less strongly than Hyg at 0.5 mg.ml^-1 as all the diploid tips had died, whereas all the haploid survived ( Table 1 , Supplementary Figure S6 ).

The results of the two sets of treatments were pooled together to increase the statistical power. It can be seen that diploid tips treated with 0.5 and 1 mg.ml^-1 Hyg respectively started to die after 7 and 5 days, whereas none of the controls had died after 15 days ( Figure 2 , Supplementary Table S6 ). The haploid tips treated with 0.5 and 1 mg.ml^-1 Hyg started to die after 5 days, whereas only one haploid control had died ( Figure 2 , Supplementary Table S6 ). This confirms that Hyg negatively affects the survival of both diploid and haploid tips, at 0.5 and 1 mg.ml^-1. For PMM, the diploid tips treated with 0.5 and 1 mg.ml^-1 respectively started to die after 7 and 5 days, whereas none of the controls had died after 15 days ( Figure 2 , Supplementary Table S6 ). The haploid tips treated with 0.5 and 1 mg.ml^-1 PMM also had an increased mortality, although lower than the diploids, as respectively five and 11 of them had died after 15 days, whereas only one control tip had died ( Figure 2 , Supplementary Table S6 ). It can therefore be concluded that PMM negatively affects the survival of both diploid and haploid tips at 0.5 and 1 mg.ml^-1, but less strongly than Hyg.

These results suggest that Gp. lemaneiformis branch tips are sensitive to both Hyg and PMM, but that higher concentrations of PMM are required to have a negative effect on survival. Hyg was therefore chosen as a selection antibiotic for transformation experiments.

To use Hyg as a selection antibiotic for future genetic transformation experiments, the suitable Hyg concentration needed for selection should be determined. The inhibitory concentration 50 (IC₅₀) was therefore calculated. The IC₅₀ measures the Hyg concentration needed for 50% loss of Gp. leamaneiformis viability. For the diploid tips, it could be determined for 5, 7, and 9 days of Hyg treatment (not for the other time points as the proportion of dead tips was either of 0 or 100%) ( Table 2 ). The IC₅₀ was 0.71 mg.ml^-1 after 5 days of Hyg treatment and fell to 0.48 mg.ml^-1 after 9 days of treatment. As the range of Hyg concentration was small, the IC₅₀ 95% intervals varied between 0.52 and 0.87 at 5 days of treatment, so the determination of the IC₅₀ was not very sensitive. For the haploid tips, IC₅₀ was 0.69 mg.ml^-1 after 5 days of Hyg treatment and 0.45 mg.ml^-1 after 7 days. A concentration of 0.5 mg.ml^-1 was finally chosen to be tested on diploids (see below), with which 50% loss of viability should be expected after 9 days and therefore 50% of survivors should be expected to be transformants.

After having selected the most suitable antibiotic and antibiotic concentration for Gp. lemaneiformis selection, transient DNA transformation was tested. A plasmid containing the Hyg resistance gene (Hygromycin phosphotranspherase or Hpt) was therefore chosen to select resistant transformed cells. The pGFPGUSPlus plasmid containing the Hpt gene, as well as the EGFP and β-Glucuronidase (GUS) reporter genes, all under the P35S promoter, was used ( Supplementary Figure S7A ). To first determine whether the pGFPGUSPlus plasmid could be successfully transformed into branch tip cells. The pGFPGUSPlus plasmid was coated on microparticles and bombarded on Gp. lemaneiformis tertiary branch tips without antibiotic selection. After having removed potential plasmid contamination outside the branch tips by DNAse treatment, PCR with primers specific for the Hpt sequence was carried out on total DNA extracted from bombarded tips, 2–8 days after bombardment. The gel electrophoresis results showed that amplification of the Hpt fragment (514 bp) was detectable in the tips from 2 to 8 days after microparticle bombardment ( Figure 3 ), although the signal was weaker after 5 days. It can hence be concluded that the pGFPGUSPlus plasmid was present in the tips and therefore that it was indeed transiently transformed in the bombarded tips, at least until 8 days after bombardment. Microparticle bombardment is thus a suitable method for transient DNA transformation in Gp. lemaneiformis under the conditions tested here. Before performing the transformation with Hyg selection, efficient expression of the Hpt gene in the tips needed to be checked, so transgene expression was verified first.

After having confirmed that the Hpt gene was expressed in the transformed tips, efficient resistance against Hyg should be tested. Two hundred tips were transformed with either the pGFPGUSPlus plasmid or with no plasmid for the control and treated with 0.5 mg.ml^-1 Hyg. At this concentration, 50% loss of viability should be expected after 9 days, and therefore 50% of survivors should be expected to be transformants ( Table 2 ). The dead tips were counted every day ( Figure 6 ). As no significant difference in survival could be observed between the tips transformed with the plasmid and the controls after 9 days, the treatment was continued during 20 days to keep the selection pressure and determine whether a different survival could be observed. The tips transformed with the plasmid only had a significant lower death rate than the control 13 days after bombardment ( Figure 6 , Supplementary Table S7 ). After 20 days, all the control tips had died. Five tips transformed with the plasmid had survived, that is, some parts of the tip’s tissue had conserved a red color, but they died shortly after having been transferred to seawater without antibiotic, so they could not be regenerated.

The experiment was repeated with 1 mg.ml^-1 Hyg instead of 0.5. Five hundred tips were bombarded with pGFPGUSPlus and 500 with uncoated gold particles, and the number of dead tips was counted every day. After 10 days, the surviving tips were transferred to normal seawater to see whether they could recover, but they died after a few days ( Figure 6 ). The survival of tips transformed with the pGFPGUSPlus plasmid was however significantly different from the control from days 6 to 10 ( Figure 6 , Supplementary Table S8 ), suggesting that the transfection of the pGFPGUSPlus plasmid in the tips had a positive impact on their survival.

The CaMV35S promoter was shown to have various efficiencies in red algae. It was indeed shown to efficiently drive gene expression in G. gracilis (Huddy et al., 2012), but it was found to have a low activity in P. yezoensis and Cyanidioschyzon merolae (Fukuda et al., 2008; Watanabe et al., 2011). This could be due to differences in the promoter structure and transcriptional regulation of protein-coding genes between red algae and dicot plants. This article showed that the CaMV35S promoter could efficiently drive the expression of the Hpt and LacZ genes in Gp. lemaneiformis, but potential higher efficiency of other promoters needed to be checked. Promoters taken from the genome of other species can usually serve as efficient heterologous promoters to drive the expression of foreign genes in algae. Porphyra’s actin promoter (PyAct1) has for instance been found effective in expression vectors in various red algae species (Uji et al., 2010; Hirata et al., 2011). The current study showed that it was also efficient in Gp. lemaneiformis but less so than the endogenous GlAct1 promoter. In conclusion, the CaMV35S, PyAct1, and GlAct1 promoters could be used alternatively for transgene expression in this species, although gene expression under the PyAct1 promoter seemed to be the weakest. CaMV35S would be more suited for stable genomic integration and expression of a transgene to avoid any risk of recombination of the GlAct1 promoter at the endogenous Actin1 locus.

Other promoters could be tried to see if gene expression could still be improved, such as promoters of other ubiquitous genes like GAPDH or Ubiquitin. The putative promoter region upstream the GAPDH and Ubiquitin GUBQ1 genes can indeed be found in the Gp. lemaneiformis genomic SRA database, and 200 bp of these regions could be cloned upstream reporter genes. However, for stable DNA integration experiments, a promoter sequence from a related species could be more favorable to avoid integration at the promoter site caused by sequence homology. GAPDH and Ubiquitin promoter sequences from G. gracilis, C. crispus or P. yezoensis could therefore be tested.

The PCR and RT-PCR results of the plasmid detection and the expression of the Hpt and mTagBFP2 genes in the transformants showed very weak PCR amplification 1 week after transformation ( Figures 3 , 4B , 7 ). This suggests that the plasmid was present in lower amounts and that the transgene was not expressed anymore or very weakly in the tips after that time, which could be due to the plasmid being diluted because of cell division, or being degraded. The antibiotic selection process should therefore be done within this timeframe of one week for future experiments.

The GUS staining and BFP observation results also confirm that microparticle bombardment is a reliable method for DNA transformation in Gp. lemaneiformis branch tips. Moreover, they show that the two reporter genes are suited for this species. The GUS staining and BFP fluorescence, however, could not be observed in many cells, indicating that transformation efficiency was not high. This may be due to a rather low efficiency of DNA coating on the gold particles or to plasmid damage during the bombardment process. Different coating conditions could be tested in the future to tackle this problem, with different buffers and DNA concentrations. The mTagBFP2 sequence could also be optimized to the codon usage of Gp. lemaneiformis to see whether the expression could be improved. The Cyan Fluorescent Protein (CFP) could also be tested, as it has been successfully used in Pyropia (Lim et al., 2013).

There was a discrepancy between the viability that was expected from the IC₅₀ results ( Table 2 ) and the observed viability after treatment with 0.5 mg.ml^-1 Hyg ( Figure 6 ). Fifty percent viability was expected for diploid tips after nine days of 0.5 mg.ml^-1 Hyg treatment ( Table 2 ), but about 80% viability was observed in both transformed and non-transformed tips ( Figure 6 ). This could be caused by the fact that a smaller number of tips were tested in the IC₅₀ experiment or by the way the dead tips were screened. As the tips that had some red pigments left were not considered dead, there could be variability among individual tips, in particular if their size varied slightly.

Resistant tips transformed with the Hpt resistance gene could still not be recovered after treatment with Hyg, suggesting either that the transformation rate was too low, or that the Hyg treatment was inadequate. It was determined that a concentration of 0.5 mg.ml^-1 Hyg was not enough to screen the tips transformed with the resistance gene from the control tips ( Figure 6 ). With a concentration of 1 mg.ml^-1, although the tips could not be regenerated, their survival was increased compared with controls between 6 and 10 days ( Figure 6 ). This suggests that the Hpt gene conferred Hyg resistance to the tips, but that the regeneration setup has to be improved. To be able to regenerate transformed tips, shortening the treatment period could for instance be tested. The RT-PCR results ( Figure 4 ) suggest that the transgene was almost not expressed anymore in the transformed tips 7 days after transformation. The 10 days Hyg treatment of tips transformed with pGFPGUSPlus ( Figure 6 ) was therefore too much, as the tip cells would probably not be able to resist against Hyg from 7 to 10 days. A shorter Hyg treatment of 7 days or less could thus be tested, and the tips could be transferred to seawater without antibiotic after the short Hyg treatment to test whether they could be regenerated. Regeneration of the tips might also be improved by increasing the oxygenation of the culture flasks.

To tackle the problem of low transformation rate, the number of individuals to be transformed could be increased, for instance at least 500 tips or more could be used for each bombardment. Second, the tips could be slightly cut with a blade before microparticle bombardment to facilitate the penetration of the coated gold particles in the tissue, as the Gp. lemaneiformis cell wall is thick. Enzymes degrading the cell wall could also be used to facilitate the transformation, such as glycoside hydrolases, polysaccharide lyases or carbohydrate esterases, which have successfully been used in green macroalga to degrade the cell wall (Costa et al., 2022). Third, transformation could be tried in spores instead of branch tips, for example, via electroporation as what was done in the unicellular alga Chlamydomonas reinhardtii (Ferenczi et al., 2017). A transformed spore could therefore give rise to a fully transformed organism. However, to obtain stably transformed organisms, strategies for obtaining stable DNA integration in the genome will have to be developed.