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Establishment of regeneration, transformation, and genome editing procedures for a seed-propagated carnation (Dianthus caryophyllus L.) variety

Establishment of regeneration, transformation, and genome editing procedures for a... Carnations (Dianthus caryophyllus L.) are amongst the three most commercially valuable cut flowers worldwide. How - ever, traditional breeding methods are often time-consuming and labor-intensive. Although genome editing is used as an alternative method for creating new varieties, the high heterozygosity of carnations inhibits the ability to maintain vari- etal characteristics in null segregants except for target-derived traits. The use of homozygous lines is a possible solution. Therefore, this study aimed to establish regeneration, transformation, and genome editing methods using seed-carnation varieties. The effects of four auxins (indole-3-butyric acid, IBA; a-naphthaleneacetic acid, NAA; 2,4-dichlorophenoxy - acetic acid, 2,4-D; and 3-indoleacetic acid, IAA) and five cytokinins (6-benzyladenine, BA; thidiazuron, TDZ; kinetin, KT; zeatin, ZT; and N -2-isopentenyl adenine, 2IP) on callus and shoot induction were evaluated. The combination of 0.05 mg/l 2,4-D and 4 mg/l TDZ had the highest shoot formation rate at 28%. In addition, shoot hyperhydricity was reduced by increasing the size of culture vessels. Sucrose, agar, and AgNO concentrations, as well as pH, were optimized to facilitate regeneration. Hygromycin at 12.5 mg/l was subsequently used as the selection agent after Agrobacterium- mediated transformation. Finally, the phytoene desaturase gene was knocked out using the CRISPR/Cas9 system. The obtained albino shoot had a one-base deletion or two-base insertion in the genome sequence. To our knowledge, this is the first study to establish a system for genome editing of callus-derived shoots from a homozygous seed-propagated carnation, which may contribute to the rapid breeding of the new varieties. Key message This is the first report to establish regeneration, transformation, and genome editing methods using homozygous seed- carnation cultivars to maintain varietal characteristics in null segregants. Keywords CRISPR/Cas9 · Stable transformation · Homozygote · Hyperhydricity · Callus induction · Shoot induction Abbreviations IBA Indole-3-butyric acid NAA a-naphthaleneacetic acid 2,4-D 2,4-dichlorophenoxyacetic acid IAA 3-indoleacetic acid, IAA BA 6-benzyladenine Communicated by Klaus Eimert. TDZ Thidiazuron KT Kinetin Yuichi Uno ZT Zeatin [email protected] 2IP N -2-isopentenyl adenine Graduate School of Agricultural Science, Kobe University, 1–1 Rokkodai, Nada-ku, Kobe 657–8501, Japan Institute of Vegetable and Floriculture Science, NARO, Tsukuba, Ibaraki 305–0852, Japan 1 3 8 Page 2 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Introduction which is a disadvantage in transformation (Zhang et al. 2010; Jha et al. 2011; Sharma et al. 2011). In carnation, some Carnation (Dianthus caryophyllus), belongs to the Caryo- studies have focused on direct regeneration (Frey and Janick phyllaceae family, originates from Mediterranean, and is 1991; Fisher et al. 1993; Watad et al. 1996), and some in currently cultivated worldwide (Anonis 1985; Reddy 2016). callus-mediated regeneration (Thakur et al. 2002; Thakur & It is among the top three commercially valuable cut flowers. Kanwar 2018; Jorapur et al. 2018). However, homozygous Carnations have been bred for improved color and shape, seed-propagated carnation genotypes have not been used as sensitivity of floral differentiation to day length and low tem - experimental materials. Another major challenge in carna- peratures, as well as resistance to pathogens. Crossbreeding, tion in vitro culture is hyperhydricity. Hyperhydric carna- natural variation, and artificially induced variation, such as tion shoots become yellow, swollen, glassy, and curved after radiation and chemical substances, are predominantly used being transferred to soil; thus, they do not survive (Yadav et to obtain new varieties (Henderson and Salt 2017). To date, al. 2003). Although callus-mediated regeneration has been crossbreeding has been the main approach used to create extensively studied, homozygous carnations have not been new carnation varieties to meet commercial demands. How- used as experimental materials (Thakur et al. 2002; Thakur ever, this methodology requires a great amount of labor and & Kanwar 2018; Jorapur et al. 2018). is time-consuming. Recently, genome editing has appeared In the current study, we targeted phytoene desaturase as a novel technology to overcome these problems, making gene (PDS) for genome editing. PDS catalyzes diapophy- possible the release of new plant genotypes with accurate toene to produce ζ-carotene in the carotenoid synthesis trait modifications in less time. Furthermore, CRISPR/Cas9 pathway. Plants often exhibit albino and dwarfing pheno - genome editing can prevent transgenesis, which may help to types when PDS is suppressed. This gene is widely used avoid public and administrative concerns about cultivation to evaluate CRISPR/Cas9 efficiency since PDS-silenced and commercialization of genetically modified crops. This plants can be easily detected (Pan et al. 2016; Hooghvorst approach has been applied on more than 10 ornamental flow - et al. 2019; Wilson et al. 2019). Therefore, we targeted the ers and is reported in three review papers (Ramirez-Torres PDS gene for genome editing in carnations. In this study, et al. 2021; Mekapogu et al. 2023; Liu et al. 2024). Regard- we established for the first time the regeneration, transfor - ing the application of gene editing techniques to carnation, mation, and genome editing of callus-derived shoots from two reports have been published until date. One described seed-propagated carnations that can consistently acquire the use of protoplasts but shoots were not obtained (Adedeji null segregants. Our finding is expected to contribute to the et al. 2024). In the other report, authors applied an electro- rapid breeding of new carnation varieties in the future. poration based approach to successfully produce mutations in the anthocyanidin synthase gene, but chimerism of the regenerated plants remained a challenge (Mori et al. 2024). Materials and methods Carnations are typically vegetatively propagated through cutting because most varieties are heterozygotes (Yagi Plant materials 2015). When heterozygotes are propagated by seed, traits segregate in the next generation and various character- Seeds of ‘Chabaud Giant’ were purchased from Fukukaen istics cannot be maintained. This is a disadvantage in the Nursery & Bulb Co., Ltd. (Aichi, Japan) and stored in a process of obtaining null segregants without foreign genes refrigerator at 4 °C until use (Fig S1). This variety blooms after genome editing. To prevent this, genome editing is with double, fringed, and medium-sized flowers, making it performed by transiently expressing foreign genes without suitable for early harvesting during outdoor cultivation. introducing them into the genome (Hamada et al. 2017; Adedeji et al. 2024) or by using homozygous lines for seed In vitro seed germination propagation. In genome editing using homozygotes, foreign genes can be removed by selfing to obtain null segregants Seeds were first surface-sterilized with 70% ethanol for with the original traits maintained. Since this material has 5 min, followed by 2% sodium hypochlorite with one drop not yet been tested in carnations, this study developed a of 10% Tween 20 for 15 min. They were then rinsed thrice genome editing system using seed-propagated variety. with sterilized distilled water. Twenty milliliters of solid To applied genome editing techniques, it is essential to medium containing Murashige and Skoog (MS) (1962) previously develop tissue culture procedures such as adven- basal salts, 30 g/l sucrose, and 8 g/l agar (FUJIFILM Wako titious shoot regeneration and transformation (Bekalu et al. Pure Chemical Corporation, Japan) were prepared and 2023). Callus-mediated (indirect) regeneration is advisable adjusted to pH 5.8 ± 0.1 before sterilization with an auto- since direct shoot regeneration often produces chimeras, clave at 121 °C for 20 min. Sterilized seeds were sown on 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 3 of 13 8 the medium in Φ90 × 19 mm Petri dishes. Seeds were cul- Rooting experiments tured in a growth chamber at 26 °C under a 16 h photoperiod provided by cool-white fluorescent lamps at a light intensity Newly induced shoots in 1.5 months after inoculation were of approximately 100 µmol/s/m . excised from explants or calli onto rooting medium at pH 5.8 supplemented with MS basal salts, 30 g/l sucrose, and Determination of the phytohormones involved in 4 g/l gellan gum, which facilitates observation of the roots callus induction better than agar. The medium included combinations of the following phytohormones: 0.01 mg/l KT, 5 mg/l GA ; Seeds sown in the medium germinated in about 3 days. 0.01 mg/l NAA, 0.01 mg/l IBA. To test the vessels, 50, 200, Subsequently, the top four true leaves were taken from each and 450 ml culture bottles were used, and the medium was seedling and cut into 5 mm square segments after 18 days filled to 40% of the vessel volume. Three shoots were placed of seedling culture. The segments were cultured in the same per bottle in triplicate. The culture environment was the medium used for germination but with phytohormones. same as that described in the former section “in vitro seed The experiment was divided into two parts. In experiment germination”. Rooting data was recorded after 1 month. 1, combinations of various hormones (IBA, NAA, 2,4-D, IAA, BA, TDZ, KT, zeatin, and 2iP) were used at 0.5 and Eec ff t of antibiotics on shoot regeneration 1.0 mg/l as callus induction medium (CIM). Each treatment combination was tested with 10 explants in triplicate. The The optimal antibiotic concentration for selection of trans- growth conditions were the same as those used for germina- formed tissues after Agrobacterium infection was deter- tion. After 1 month, the obtained calli were moved to a shoot mined. Kanamycin at 25, 50, 75, or 100 mg/l and hygromycin induction medium (SIM) supplemented with 0.1 mg/l NAA at 5.0, 7.5, 10.0, 12.5, or 15 mg/l were tested with a com- and 1.0 mg/l BA as described by Nontaswatsri et al. (2004). bination of 0.5 mg/l 2,4-D and 4.0 mg/l TDZ. All the other After 0.5 months, shoot induction rates were recorded, dis- components in the medium were the same as those used for tinguishing the CIM origin of the callus. 2,4-D as auxin seeds germination. Leaves of ‘Chabaud Giant’ were used and TDZ as cytokinin, which had high final shoot induc - as explants for culture under the same conditions as those tion rates, were selected as the hormones for CIM, and the described in the former section “in vitro seed germination”. detailed concentrations were reexamined as Experiment 2. Each treatment combination was tested with 10 explants in The combinations of 2,4-D and TDZ were examined at con- triplicate. The formation of callus and shoot were observed centrations of 0.01, 0.05, 0.1, 1.0, 2.0, 4.0, and 8.0 mg/l. The after 1 month. medium pH was adjusted to 5.8 ± 0.1 before sterilization at 121 °C for 20 min. The culture environment was the same as Cloning of DcPDS that described in the subsection above. The segments were cultured for 1.5 months. In addition, each treatment com- Genomic DNA of ‘Chabaud Giant’ was extracted from bination was tested with 10 explants in triplicate. Because leaves using the Nucleon PhytoPure Plant DNA Extract Kit this hormone combination yielded callus followed by shoot, (Cytiva, UK). Specific primers were designed based on the no independent SIM trials were conducted in Experiment 2. gene fragment in the carnation ‘Francesco’ database (Yagi et al. 2014). PDS genes were found in the Carnation DB Eec ff t of other factors in the induction of healthy and named DcPDS1, DcPDS2, and DcPDS3. Because the calli and shoots DcPDS2 and DcPDS3 sequences were identical, we later named them DcPDS2/3. We designed primers for the mid- Additional factors such as sucrose (20, 30, 40, and 50 g/l), dle and back regions of DcPDS1 and for the front and back agar (4, 8, 12, and 16 g/l), AgNO (0.5, 1.0, and 2.0 mg/l), regions of DcPDS2/3 (Table S1). The PCR reaction system and pH (5.0, 5.4, 5.8, and 6.2), were studied. The different was as follows: 10× PCR Buffer containing 5 µl KOD Plus treatments were compared with standard conditions based Neo, 5 µl dNTPs, 3 µl MgSO , 1 µl upstream primer, 1 µl on pH 5.8, as well as 8 g/l agar, 30 g/l sucrose, 0.05 mg/l downstream primer, 1 µl KOD plus Neo, 200 ng template 2,4-D, and 4 mg/l TDZ. Culture conditions were similar to DNA, and ddH O to complete the final volume. The reac - those described for seed germination. Each treatment com- tion conditions were as follows: 94 °C for 2 min; 98 °C for bination was tested with 10 explants in triplicate. 10 s, (Tm) °C for 30 s, and 68 °C for 30 s/kb, 45 cycles; and then hold at 25 °C. The PCR products were detected through 1.2% agarose gel electrophoresis. PCR product purification was performed using the GEL/PCR Purification Mini Kit (FAVORGEN) according to the manufacturer’s 1 3 8 Page 4 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 instructions. Next, purified PCR products were cloned with Biosystems), SeqStudio, or SeqStudio Flex 8 (Thermo the pTA2 vector, transformed into Escherichia coli DH5α, Fisher Scientific) automated DNA sequencing system using and identified via PCR. The positive clone was then selected the Prism Ready Reaction DyeDeoxy Terminator Cycle for sequencing. Sequencing kit 3.1 (Applied Biosystems Division, Perkin- Elmer, Foster City, CA, USA). Editing of genome sequence Construction of the CRISPR/Cas9 vector was confirmed using the CLC Main Workbench Version 20 sequence analysis programs (QIAGEN Aarhus A/S, Aarhus, Comparison of the PDS amino acid sequence of the carna- Denmark). tion with that of other plants revealed the first conserved domains in the second and fourth exons of DcPDS1 and DcPDS2/3, respectively (Fig S2). To knock out all three Results genes, 20 nt gRNAs were designed adjacent to the PAM sequence (Fig S3). Primers containing the AarI restric- Callus and shoot induction with different hormone tion site were annealed and ligated into pKIR1.1 (Tsutsui combinations and Higashiyama 2017). pKIR1.1 was a gift from Tetsuya Higashiyama (Addgene plasmid # 85758; h t t p : / / n 2 t . n e t / a d In the first experiment, four types of auxins were used in d g e n e : 8 5 7 5 8 ; RRID: Addgene_85758). A vector was con- combination with five cytokinins to select the appropriate structed and verified through standard cloning and sequenc - phytohormone combination for the induction of calli and ing, respectively. shoots from explants (Table 1; Fig S4). KT and IBA did not induce callus formation at most concentrations. Similarly, Transformation by Agrobacterium infection combinations of IAA with BA, KT, or 2IP did not induce callus hardly at all. The other 16 combinations induced calli Agrobacterium LBA4404 was used for the transformation efficiently, with induction rates ranging from 50.0 to 100%. experiments. Agrobacterium harboring the vector construc- However, many calli were either too small or hyperhydric tion for CRISPR/Cas9 was cultured in LB liquid medium (Fig S5A; S5B). Some of them induced roots during one supplemented with 100 mg/l rifampicin and 100 mg/l spec- month of culture in CIM (Fig S4), but none of them induced tinomycin at 28 °C for 2 d. A large-scale culture was per- shoots. Therefore, the callus was transplanted to the previ- formed until OD ≥ 0.6. The centrifuged pellet was then ously reported SIM supplemented with 0.1 mg/l NAA and adjusted to OD = 0.6–0.7 using fresh LB liquid medium 1.0 mg/ BA (Nontaswatsri et al. 2004). However, it was dif- supplemented with 0.1 µM acetosyringone and mercapto- ficult to obtain shoots from calli cultured in this SIM. In ethanol. The leaf segments were soaked in Agrobacterium 0.5 month after transplanting, shoots only grew from the suspension for 7 min, dried with filter paper, and placed onto two calli cultured in a medium supplemented with 0.5 mg/l MS solid medium supplemented with 0.05 mg/l 2,4-D and 2,4-D and 1.0 mg/l TDZ (Fig S5C). 4.0 mg/l TDZ. All the other components in the medium, and culture conditions were the same as those used for seeds ger- Callus induction with 2,4-D and TDZ mination. They were then co-cultivated for 3 d and moved to the selection medium containing antibiotics; 12.5 mg/l In the experiment 1 in the previous chapter, shoots were hygromycin and 50 mg/l meropenem. Each treatment com- induced only on media containing 2,4-D and TDZ. There- bination was tested with 10 explants in 80 replicates. The fore, as experiment 2, combinations of these phytohormones medium was changed every 2 weeks. Regenerated albino were tested at various concentrations to improve shoot for- shoots were selected as candidate genome-edited plants for mation efficiency. Callus formation was observed at all con - further analysis. centrations but decreased at 2,4-D concentrations > 1.0 mg/l (Fig. 1A). At a 2,4-D concentration > 2.0 mg/l, most calli Confirmation of the edited genome sequence were small, pale yellow, and became hyperhydric with- out shoot formation (Fig. 1A). The best conditions for the To determine the genome editing efficiency of DcPDSs, the induction of healthy green calli with shoots were 0.05 mg/l target sequence was amplified from gDNA using PCR and 2,4-D and 4.0 mg/l TDZ (Fig. 1B, Table S2). cloned into the standard sequencing vector (pSKII ) using an In-Fusion HD Cloning Kit (Takara Bio). The positive Eec ff t of other factors in callus and shoot induction clones were selected for Sanger sequencing. The nucleotide sequences were determined using the dideoxy chain-termi- To improve the shoot induction medium from the previ- nation method on 3130, 3130xl Genetic Analyzer (Appllied ous conditions (30 g/l sucrose, 8 g/l agar, pH = 5.8, absence 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 5 of 13 8 Table 1 Callus induction rates (%) from carnation leaf explants under differ ent phytohormone combinations Auxin (1.0 mg/l) Mean IBA NAA 2,4-D IAA Cytokinin BA 72.2 ± 20.0a 72.2 ± 20.0a 83.3 ± 9.6a 0.0 ± 0.0c 56.9 ± 11.9mn (0.5 mg/l) TDZ 72.2 ± 14.8a 77.8 ± 22.2a 72.2 ± 5.6a 83.3 ± 9.6a 76.4 ± 6.3mn KT 0.0 ± 0.0c 50.0 ± 19.2abc 88.9 ± 11.1a 0.0 ± 0.0c 34.7 ± 12.2n ZT 66.7 ± 9.6ab 100.0 ± 0.0a 88.9 ± 11.1a 77.8 ± 11.1a 83.3 ± 5.4 m 2IP 94.4 ± 5.5a 77.8 ± 11.1a 100.0 ± 0.0a 5.6 ± 5.6bc 69.4 ± 11.8mn Mean 61.1 ± 9.7xy 75.6 ± 7.6x 86.7 ± 4.0x 33.3 ± 10.7y Auxin (0.5 mg/l) Mean IBA NAA 2,4-D IAA Cytokinin BA 88.9 ± 5.6a 72.2 ± 20.0ab 100.0 ± 0.0a 0.0 ± 0.0d 65.2 ± 12.6mn (0.5 mg/l) TDZ 94.4 ± 5.6a 94.4 ± 5.6a 88.9 ± 5.6a 88.9 ± 5.6a 91.7 ± 2.5 m KT 5.6 ± 5.6d 61.1 ± 14.7abc 100.0 ± 0.0a 11.1 ± 5.6d 44.4 ± 12.2n ZT 88.9 ± 5.6a 94.4 ± 5.6a 94.4 ± 5.6a 27.8 ± 5.6 cd 76.4 ± 8.9mn 2IP 94.4 ± 5.6a 77.8 ± 14.7a 94.4 ± 5.6a 33.3 ± 9.6bcd 75.0 ± 8.6mn Mean 74.4 ± 9.5x 80.0 ± 6.1x 95.6 ± 2.0x 32.2 ± 8.5y Auxin (0.5 mg/l) Mean IBA NAA 2,4-D IAA Cytokinin BA 55.6 ± 5.6ab 88.9 ± 9.6a 88.9 ± 11.1a 0.0 ± 0.0c 58.3 ± 11.3 nm (1.0 mg/l) TDZ 94.4 ± 11.1a 100.0 ± 0.0a 83.3 ± 9.6a 77.8 ± 5.6a 88.9 ± 3.8 m KT 11.1 ± 11.1bc 72.2 ± 14.7a 83.3 ± 9.6a 0.0 ± 0.0c 41.7 ± 11.9n ZT 94.4 ± 5.6a 83.3 ± 9.2a 83.3 ± 9.6a 66.7 ± 1.7a 81.9 ± 5.6 m 2IP 94.4 ± 5.6a 94.4 ± 5.6a 88.9 ± 5.6a 5.6 ± 5.6c 70.8 ± 11.6mn Mean 70.0 ± 9.2x 87.8 ± 4.1x 85.6 ± 3.6x 30.0 ± 9.8y Data are expressed as the mean rates (%) of callus induction rates ± SE (n = 6, r = 3) after 1 month. Data followed by die ff rent letters are signifi - cantly die ff rent ( P < 0.05), as determined using Tukey–Kramer’s honestly signic fi ant die ff rence (HSD) test of AgNO ), various concentrations of sucrose, agar and Therefore, it was not necessary to include AgNO in the 3 3 AgNO , and pH values were investigated. The shoot induc- medium at the concentrations tested. tion rates of the base conditions were compared in the 2,4-D and TDZ concentration tests and in the tests of the other Root induction by different hormones and vessels components. Callus and shoot induction rates did not sig- nificantly differ when cultured at different concentrations Different hormones and vessels were evaluated to increase of sucrose or agar and under varying pH values, however, the rooting rate and reduce hyperhydricity. In 200 ml ves- hyperhydricity was variable (Table 2). In the sucrose treat- sels with GA fixed at 5 mg/l, different combinations of ment, rate of hyperhydric calli was significantly higher at a three phytohormones (NAA, KT, and IBA) at various con- concentration of 20 g/l. Based on the photos, 50 g/l sucrose centrations did not significantly affect in rooting rate from resulted in yellowish, brown, and fragile calli (Fig S6A). shoots and shoot hyperhydricity rate (Table 3). However, Agar tended to increase hyperhydricity at lower concentra- comparison of different volume vessels with fixing 5.0 mg/l tions, with 4 g/l being significantly lower than with 12 and GA and 0.01 mg/l IBA revealed that rates of rooted shoot 16 g/l (Table 2). Calli were dehydrated and their surface was the lowest significantly in 50 ml bottles, less than half appeared white at > 12 g/l agar (Fig S6B). Hyperhydricity of the 200 ml and 450 ml. In addition, the rate of hyperhy- tended to increase with advancing acidity and alkalinity, dric plantlets decreased with an increase in vessel volume. such as 5.0 and 6.2, and was significantly lower at 5.4 and The rate of hyperhydric plantlets was 22% and 90% in 450 5.8 than at 5.0. At pH 5.0, there were many light green calli and 50 ml bottles, respectively (Table 3). Based on these (Fig S6C). These results suggest that the base conditions of results, phytohormones were not critical, and larger vessel 30 g/l sucrose, 8 g/l agar, pH = 5.8 are adequate to reduce volumes, such as 450 ml, were more effective in maintain - hyperhydricity. As the concentration of AgNO increased, ing healthy shoots and promoting rooting. Some rooted the callus induction rate decreased (Table 2). In the AgNO - regenerants were successfully acclimated and potted in soil supplemented medium, callus hyperhydricity was sup- (Fig. 2). Through the above experiments, the following con- pressed, but shoots were not induced (Table 2; Fig.S6D). ditions were determined to obtain regenerants from seed- propagated carnations. For both callus and shoot induction, 1 3 8 Page 6 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Fig. 1 Effects of different concentration combinations of 2,4-D and explants inducing shoots at various concentrations of 2,4-D and TDZ TDZ on callus/shoot formation from carnation leaf segments. Photo- (B). Ten leaf segments were incubated as explants in each Petri dish graphs of calli/shoots in medium supplemented with 2,4-D and TDZ at (n = 10, r = 3). Photographs were taken 1 month after inoculation different concentrations ( A). Significant differences in the number of 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 7 of 13 8 Table 2 Callus / shoot induction rates under different medium compo - sitions other than phytohormones Treatment Callus induction Shoot induc- Rates of rates (%) tion rates (%) hyperhydric calli (%) Sucrose 20 86.7 ± 13.3 a 0.0 ± 0.0 a 53.3 ± 3.3 a (g/l) 30 96.7 ± 3.3 a 7.7 ± 3.3 a 13.3 ± 3.3 b 40 96.7 ± 3.3 a 3.3 ± 3.3 a 13.3 ± 3.3 b 50 96.7 ± 3.3 a 6.7 ± 6.7 a 16.7 ± 3.3 b Agar (g/l) 4 90.0 ± 5.8 a 10.0 ± 6.8 a 56.7 ± 23.3 a 8 96.7 ± 3.3 a 7.7 ± 3.3 a 20.0 ± 11.2 ab 12 96.7 ± 3.3 a 0.0 ± 0.0 a 0.0 ± 0.0 b 16 93.3 ± 3.3 a 7.7 ± 3.3 a 0.0 ± 0.0 b pH 5.0 90.0 ± 10.0 a 0.0 ± 0.0 a 43.3 ± 6.7 a 5.4 73.3 ± 17.6 a 0.0 ± 0.0 a 23.3 ± 3.3 b 5.8 93.3 ± 7.7 a 3.3 ± 3.3 a 16.7 ± 3.3 b 6.2 83.3 ± 12.0 a 0.0 ± 0.0 a 26.7 ± 3.3 ab AgNO 0.5 93.3 ± 6.7 a 0.0 ± 0.0 a 0.0 ± 0.0 a (mg/l) 1.0 76.7 ± 12.0 ab 0.0 ± 0.0 a 0.0 ± 0.0 a 2.0 40.0 ± 11.6 b 0.0 ± 0.0 a 0.0 ± 0.0 a Data are expressed as the mean rates (%) of callus / shoot induc- tion ± SE (n = 10, r = 3) after 1.5 months. Data followed by die ff r - ent letters are signic fi antly die ff rent ( P < 0.05), as determined using Tukey–Kramer’s HSD test leaf segments are placed on MS medium containing 30 g/l sucrose and 8 g/l agar at pH 5.8 as the basal medium, sup- Fig. 2 Appearance of regenerated carnation plantlets. Regenerants were obtained by inducing calli and shoots from leaf segments and plemented with 0.05 mg/l 2,4-D and 4.0 mg/l TDZ in petri rooting using established protocols. Healthy shoots (A) but not hyper- dishes. For rooting, shoots are grown on the basal medium hydric shoots (B) were acclimatized and transplanted into pots (C) containing the appropriate phytohormones (e.g., 5.0 mg/l GA and 0.01 mg/l IBA) in 450 ml culture bottles. screening. The antibiotic kanamycin was initially used to Genome editing using Agrobacterium-mediated select transformed shoots; however, many of the obtained transformation shoots were only non-transformants in white color. There- fore, hygromycin was selected based on the results of a pre- As we applied Agrobacterium-mediated transformation to vious study (Nontaswatsri et al. 2004). the regeneration system described in the previous section, When designing gRNA for the target DcPDS gene, it is we examined the antibiotic conditions for selecting trans- important to avoid the mutation sequence based on varietal formants. Calli were induced with < 50 mg/l kanamycin differences between the ‘Chabaud Giant’ material vari - or 10.0 mg/l hygromycin after 1 month of leaf segment ety and the ‘Francesco’ variety used for reference genome culture (Fig. S7A; S7B). Accordingly, 75 mg/l kanamycin sequences. In this study, we use PCR cloning to obtain two and 12.5 mg/l hygromycin were used for post-infection genome sequences for the DcPDS of ‘Chabaud Giant’. The Table 3 Rates of carnation shoots forming roots under different culture cond itions Phytohormone (mg/l) Vessel volume Rates of rooted shoots (%) Rates of hyperhydric shoots (%) (ml) GA NAA KT IBA 5.0 0.01 0.01 200 77.8 ± 11.1a 55.6 ± 11.1ab 5.0 0.5 0.5 200 66.7 ± 0.0ab 66.7 ± 19.3ab 5.0 0.5 200 66.7 ± 0.0ab 77.8 ± 11.1ab 5.0 0.01 50 33.3 ± 0.0b 89.9 ± 11.1a 5.0 0.01 200 77.8 ± 11.1a 55.6 ± 11.1ab 5.0 0.01 450 77.8 ± 11.1a 22.2 ± 11.1b Mean 66.7 ± 4.7 59.2 ± 6.4 Data are expressed as the mean rates of carnation shoots forming roots ± SE (n = 3, r = 3) after 1 month. Data followed by die ff rent letters are signic fi antly die ff rent ( P < 0.05), as determined using Tukey–Kramer’s HSD test 1 3 8 Page 8 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 resulting DNA sequences encoded both partial DcPDS1 ‘Chabaud Giant’ in this study. Calli were induced by most and full-length DcPDS2 or DcPDS3 (hereafter referred hormone combinations. However, particularly low levels to as DcPDS2/3). The lengths of the cloned DcPDS1 and of callus induction were observed when the auxin IAA was DcPDS2/3 were 2471 bp and 4337 bp, respectively (Fig. used (Table 1). IAA is a naturally produced plant hormone S8). The conserved regions of DcPDS1 and DcPDS2/3 that is rapidly degraded by many tissues and often cannot were designed for gRNA (Fig. S2). Albino shoots were pro- support the growth of cultured plant tissues (Hangarter et al. duced from the explants after infection with Agrobacterium 1980). In addition, the light provided by cool-white, fluores - containing the pKI1.1R vector (Fig. 3), although the shoot cent bulbs can promote the degradation of IAA. Therefore, production rate was only 2.5%. Sequence analyses of the IBA, NAA, and 2,4-D are more commonly used than IAA in albino shoots PCR amplicons revealed two different indels; plant tissue culture (Nissen and Sutter 1990; Stasinopoulos the deletion of one base (A) or the insertion of two bases and Hangarter 1990). Although the growth rate of calli was (AT) before the PAM site. Therefore, both indels provoked high under many hormone combinations, shoots were only a shift in the reading frame and a non-functional protein obtained from calli grown in a medium containing 2,4-D (Fig. 4). The probability of genome-edited shoots per regen- and TDZ. Furthermore, many calli were especially small erated shoots was 6.7%. Since the sequencing was limited and considerably hyperhydric, which may be the main rea- to albino shoots, it is possible that the normal shoots with son shoots were not obtained. Although 2,4-D is effective non-silenced PDS gene by in-frame editing were missed. If in callus induction (Maheshwari et al. 2011; Motte et al. that possibility is included, the true efficiency may be some - 2011), it is not typically effective in shoot induction (Han - what higher. garter et al. 1980). However, 2,4-D induced shoot regenera- tion in the present study, possibly because of the specificity among species or varieties. In shoot induction from carna- Discussion tion leaves, NAA is the most commonly used auxin (Frey and Janick 1991; Nakano et al. 1994; Abu-Qaoud, 2013), Tissue culture is an important step in the transgene, neces- with some use of IBA and IAA (Nontaswatsri et al. 2002; sitating the establishment of an induction protocol of callus Kanwar and Kumar, 2009). In carnations, TDZ has been and shoots (De Filippis 2014). Thus, combinations of four successfully used as a cytokinin for shoot induction from auxins and five cytokinins were evaluated to establish an leaves. With different varieties from ‘Chabaud Giant’, 0.1- in vitro regeneration protocol for homozygous carnations 4.0 mg/l of TDZ marked high shoot induction rates ranging Fig. 3 Appearance of plantlets or calli of carnation after genome plantlets without genome editing (A). Plantlets regenerated following editing. Regenerants were obtained from Agrobacterium-infected successful genome editing using the pKI1.1R vector containing the leaf fragments using established protocols. The carnation phytoene hygromycin resistance gene (B) desaturase gene (DcPDS) was targeted. Photograph show regenerated 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 9 of 13 8 Fig. 4 Altered nucleotide sequence of DcPDS by CRISPR/Cas9- than multiples of three cause frameshifts and the appearance of an derived genome editing. Electropherograms were obtained by sequenc- early termination codon or different amino acid. The sequences for ing PCR-cloned genome fragments for the wild-type (WT) and two gRNA and PAM are highlighted in yellow and light blue, respectively genome-edited strains (DcPDS1-KO and DcPDS23-KO). Indels other from 53 to 93% (Frey and Janick 1991; Nontaswatsri et al. the present study. Agar above 12/g drastically reduced the 2002; Abu-Qaoud, 2013). The concentrations used in this hyperhydricity rate (Table 2). However, the surface of the study were also in the range, suggesting that carnations are callus turned white and appeared to be low in water content, generally responsive to TDZ. inhibiting growth (Fig. S4). Previous studies have reported Different concentrations of sucrose and agar, as well that high concentrations of agar were less likely to induce as different pH values, were tested to obtain high-quality hyperhydricity, but were detrimental to shoot regeneration calli, reduce hyperhydricity, and improve shoot induction and inhibited growth, which supported our results (Arnold efficiency. Although the results showed no significant dif - and Eriksson 1984; Kadota et al. 2001; Suthar et al. 2011). ferences in shoot induction efficiency, they provided a basis The calli appeared more hyperhydric at pH 5.0 (Table 2; for selecting a more suitable medium. Shoot regeneration Fig. S6C). pH can affect the hardness of the agar medium: a occurred when the concentrations of supplemented sucrose lower pH softens the medium, whereas a higher pH hardens were more than 30 g/l (Table 2). Calli showed obvious hyper- it (Huang et al. 1995). Although shoots only grew at pH 5.8 hydricity in the medium supplemented with 20 g/l sucrose, in other media, there was no significant effect on the induc - and browning was observed when the sucrose concentra- tion rate or appearance of calli and shoots. Hyperhydricity tion was > 40 g/l. This result is similar to that observed in was thought to be due to a reduction in the amount of cel- previous research showing that increasing concentrations lulose and lignin required for cell wall lignification caused of sucrose can reduce hyperhydria, and that high sucrose by ethylene (Keverse et al. 1984). Silver ions can bind to concentrations can promote callus browning (Khosroushahi ethylene receptors and prevent the transduction pathway et al. 2011; Liu et al. 2017; Liang et al. 2019). Therefore, a (Gao et al. 2017). Therefore, AgNO has been used as an sucrose concentration of 30 g/l was the most appropriate in anti-hyperhydric agent in several plant species (Mayor et 1 3 8 Page 10 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 al. 2003; Vinoth and Ravindhran 2015). This study actually and sugarcane (Rastogi et al. 2018). Therefore, kanamycin showed that AgNO -supplemented medium suppressed cal- was unfavorable for the screening of PDS knockouts in lus hyperhydricity, but did not induce shoots unfortunately carnations. (Table 2; Fig. S6D). The carnation ‘Chabaud Giant’ used in Two studies on genome editing in carnations have this study may be too sensitive to AgNO , as even 0.5 mg/l recently been issued. One is a transient system using the AgNO was toxic too shoot differentiation. method of direct delivery of preassembled CRISPR/Cas9 Various hormones and vessels were evaluated to deter- ribonucleoproteins (RNPs) using protoplasts (Adedeji et al. mine the optimal rooting conditions. There was no signifi - 2024). Their method is superior in two aspects: elimination cant difference in rooting in the media containing different of chimerism and foreign gene-free, but organogenesis from phytohormones (Table 3). A few examples of rooting from protoplasts is difficult and shoots were not obtained. Another carnation shoots have been reported to be effective with study used a method of introducing RNPs into stem sections 1.0 mg/l IBA (Lu et al. 2021; Zheng et al. 2020) and 1.0 mg/l by electroporation (Mori et al. 2024). With this method, also NAA (Karami and Kordestani 2007; Ali et al. 2008; Zheng foreign DNA-free, they obtained regenerated shoots, but the et al. 2020). Although we did not test the effects of these problem of chimerism remained. We successfully devel- hormones alone in this experiment, it is likely that a num- oped a genome-editing system with a seed propagation-type ber of different auxins were effective to promote rooting carnation in which PDS was knocked out. Genome editing in carnations. The use of a large size vessels showed the of seed-propagated varieties established in this study will positive effect on root growth and reduced hyperhydricity accelerate carnation breeding as it will overcome the prob- (Table 3). It has been reported that nutrient supply increases lems of chimerism, persistence of foreign genes and trait and air ventilation was improved, all of which were condu- segregation, which are problems in vegetatively propagated cive to root growth and reduced hyperhydicity (Lai et al. varieties. 2005; Lynch et al. 2012). Some studies have shown that both ethylene and CO are accumulated in a closed vessel (reviewed by Polivanova and Bedarev 2022). In addition, Conclusions the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene (ACO) involving ethylene synthesis, has been In this study, a system for shoot regeneration from leaf shown to be significantly increased in hyperhydric plants. In explants of the homozygous carnation ‘Chabaud Giant’ was a well-ventilated environment, the accumulation of ethylene established. Of all the treatments tested, the best results and CO can be reduced to inhibit the hyperhydricity. of regeneration rate were reached 27.8% in MS medium Shoot induction efficiency was reduced following Agro- supplemented with 0.05 mg/l 2,4-D, 4.0 mg/l TDZ, 30 g/l bacterium infection, possibly because of altered levels of sucrose and 8 g/l agar and adjusted to pH 5.8 after 1.5 auxin and other hormones (Bettini et al. 2010; Mashigu- month. Moreover, the use of a large size vessels with 450 ml chi et al. 2019). The selection of knockout shoots for the volume was the best condition for root growth and reducing PDS gene by the antibiotic kanamycin was not success- hyperhydricity. Additionally, an Agrobacterium-mediated ful. This could be because it was difficult to recognize the transformation procedure was developed to obtain trans- difference in white color between the drug effect of kana - formed plants using hygromycin as the selection antibiotic. mycin and the loss-of-function effect of PDS. Practically Gene editing (CRISPR/Cas9) combined with the transfor- many white-colored regenerants were obtained as escapes mation/regeneration system was investigated using PDS as on the medium containing 75 mg/l kanamycin (Fig. S7C). the target gene. Sequencing analyses of the edited albino This indicates that carnations may be somewhat resistant to shoot revealed two different indels: one base deletion or kanamycin and shoots on the selection medium grow to a two bases insertion before the PAM sequence. These results certain stage. The calli on the medium containing kanamy- demonstrate that the carnation genome editing system was cin were transparent yellow (Fig. S7D), whereas those with established for the first time with a seed-propagation vari - hygromycin were healthy (green) (Fig. S7E). The results ety, which will provide convenience for the future study of show that even in the presence of antibiotic concentrations carnation gene function and the efficient breeding of new that weaken the explant, false-positive callus differentia - varieties. tion occurs with kanamycin, whereas it is suppressed with Supplementary Information The online version contains hygromycin. Albinism in non-transformed plantlets due to supplementary material available at h t t p s : / /d o i. o r g / 1 0 . 1 0 0 7 / s 1 1 2 4 0 - 0 kanamycin has been also reported in many plant species, 2 4 - 0 2 9 2 9 - 9 . such as Brassica juncea (Gao et al. 2020), Vigna mungo (Saini et al. 2003), Indian cowpea (Chaudhury et al. 2007) Acknowledgements The authors would like to thank Dr. Hiroshi Fukayama and Dr. Keiji Nishida for their advice on genome editing experiments. The authors thank Kaya Okamoto, Haruka Konishi, 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 11 of 13 8 Satoshi Kawamoto and Nanako Fukushima for their technical assis- genome editing. Int J Mol Sci 24(15):11920. h t t p s : / /d o i. o r g / 1 0 . 3 tance. The authors also thank Dr. Takako Narumi, Dr. Michio Kanechi, 3 9 0 / i j m s 2 4 1 5 1 1 9 2 0 and Dr. Hiroyasu Yamaguchi for their valuable discussions. Bettini P, Baraldi R, Rapparini F et al (2010) The insertion of the Agro- bacteriumrhizogenes rolC gene in tomato (Solanum lycopersicum L.) affects plant architecture and endogenous auxin and abscisic Author contributions ZJL performed tissue culture, Agrobacterium- acid levels. Sci Hortic 123(3):323–328. h t t p s : / /d o i. o r g / 1 0 . 1 0 1 6 / j mediated transformation, cloning, and sequencing of DcPDS genes; . s c i e n t a . 2 0 0 9 . 0 9 . 0 1 3 generated knockout transformants via genome editing; and drafted the Chaudhury D, Madanpotra S, Jaiwal R et al (2007) Agrobacterium manuscript. YU designed the experiments, analyzed the data, and re- tumefaciens-mediated high frequency genetic transformation of vised the manuscript. MY and RK analyzed the data and revised the an Indian cowpea (Vigna unguiculata L. Walp.) Cultivar and trans- manuscript. All the authors have read and approved the final manu - mission of transgenes into progeny. Plant Sci 172(4):692–700 script. De Filippis LF (2014) Crop improvement through tissue culture. Improvement of crops in the era of climatic changes. Springer, Funding Open Access funding provided by Kobe University. New York. https://doi.or g/10.10 07/97 8-1-4614-8830-9_12 This project was supported by JST SPRING (Grant No. JPMJFS2126) Fisher M, Ziv M, Vainstein A (1993) An efficient method for adventi - and partially supported by JSPS KAKENHI (grant Nos. 17K07654 tious shoot regeneration from cultured carnation petals. Sci Hortic and 23K05214). 53(3):231–237. https://doi.or g/10.10 16/03 04-4238(93)90071-W Frey L, Janick J (1991) Organogenesis in carnation. 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Establishment of regeneration, transformation, and genome editing procedures for a seed-propagated carnation (Dianthus caryophyllus L.) variety

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Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2024
ISSN
0167-6857
eISSN
1573-5044
DOI
10.1007/s11240-024-02929-9
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Abstract

Carnations (Dianthus caryophyllus L.) are amongst the three most commercially valuable cut flowers worldwide. How - ever, traditional breeding methods are often time-consuming and labor-intensive. Although genome editing is used as an alternative method for creating new varieties, the high heterozygosity of carnations inhibits the ability to maintain vari- etal characteristics in null segregants except for target-derived traits. The use of homozygous lines is a possible solution. Therefore, this study aimed to establish regeneration, transformation, and genome editing methods using seed-carnation varieties. The effects of four auxins (indole-3-butyric acid, IBA; a-naphthaleneacetic acid, NAA; 2,4-dichlorophenoxy - acetic acid, 2,4-D; and 3-indoleacetic acid, IAA) and five cytokinins (6-benzyladenine, BA; thidiazuron, TDZ; kinetin, KT; zeatin, ZT; and N -2-isopentenyl adenine, 2IP) on callus and shoot induction were evaluated. The combination of 0.05 mg/l 2,4-D and 4 mg/l TDZ had the highest shoot formation rate at 28%. In addition, shoot hyperhydricity was reduced by increasing the size of culture vessels. Sucrose, agar, and AgNO concentrations, as well as pH, were optimized to facilitate regeneration. Hygromycin at 12.5 mg/l was subsequently used as the selection agent after Agrobacterium- mediated transformation. Finally, the phytoene desaturase gene was knocked out using the CRISPR/Cas9 system. The obtained albino shoot had a one-base deletion or two-base insertion in the genome sequence. To our knowledge, this is the first study to establish a system for genome editing of callus-derived shoots from a homozygous seed-propagated carnation, which may contribute to the rapid breeding of the new varieties. Key message This is the first report to establish regeneration, transformation, and genome editing methods using homozygous seed- carnation cultivars to maintain varietal characteristics in null segregants. Keywords CRISPR/Cas9 · Stable transformation · Homozygote · Hyperhydricity · Callus induction · Shoot induction Abbreviations IBA Indole-3-butyric acid NAA a-naphthaleneacetic acid 2,4-D 2,4-dichlorophenoxyacetic acid IAA 3-indoleacetic acid, IAA BA 6-benzyladenine Communicated by Klaus Eimert. TDZ Thidiazuron KT Kinetin Yuichi Uno ZT Zeatin [email protected] 2IP N -2-isopentenyl adenine Graduate School of Agricultural Science, Kobe University, 1–1 Rokkodai, Nada-ku, Kobe 657–8501, Japan Institute of Vegetable and Floriculture Science, NARO, Tsukuba, Ibaraki 305–0852, Japan 1 3 8 Page 2 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Introduction which is a disadvantage in transformation (Zhang et al. 2010; Jha et al. 2011; Sharma et al. 2011). In carnation, some Carnation (Dianthus caryophyllus), belongs to the Caryo- studies have focused on direct regeneration (Frey and Janick phyllaceae family, originates from Mediterranean, and is 1991; Fisher et al. 1993; Watad et al. 1996), and some in currently cultivated worldwide (Anonis 1985; Reddy 2016). callus-mediated regeneration (Thakur et al. 2002; Thakur & It is among the top three commercially valuable cut flowers. Kanwar 2018; Jorapur et al. 2018). However, homozygous Carnations have been bred for improved color and shape, seed-propagated carnation genotypes have not been used as sensitivity of floral differentiation to day length and low tem - experimental materials. Another major challenge in carna- peratures, as well as resistance to pathogens. Crossbreeding, tion in vitro culture is hyperhydricity. Hyperhydric carna- natural variation, and artificially induced variation, such as tion shoots become yellow, swollen, glassy, and curved after radiation and chemical substances, are predominantly used being transferred to soil; thus, they do not survive (Yadav et to obtain new varieties (Henderson and Salt 2017). To date, al. 2003). Although callus-mediated regeneration has been crossbreeding has been the main approach used to create extensively studied, homozygous carnations have not been new carnation varieties to meet commercial demands. How- used as experimental materials (Thakur et al. 2002; Thakur ever, this methodology requires a great amount of labor and & Kanwar 2018; Jorapur et al. 2018). is time-consuming. Recently, genome editing has appeared In the current study, we targeted phytoene desaturase as a novel technology to overcome these problems, making gene (PDS) for genome editing. PDS catalyzes diapophy- possible the release of new plant genotypes with accurate toene to produce ζ-carotene in the carotenoid synthesis trait modifications in less time. Furthermore, CRISPR/Cas9 pathway. Plants often exhibit albino and dwarfing pheno - genome editing can prevent transgenesis, which may help to types when PDS is suppressed. This gene is widely used avoid public and administrative concerns about cultivation to evaluate CRISPR/Cas9 efficiency since PDS-silenced and commercialization of genetically modified crops. This plants can be easily detected (Pan et al. 2016; Hooghvorst approach has been applied on more than 10 ornamental flow - et al. 2019; Wilson et al. 2019). Therefore, we targeted the ers and is reported in three review papers (Ramirez-Torres PDS gene for genome editing in carnations. In this study, et al. 2021; Mekapogu et al. 2023; Liu et al. 2024). Regard- we established for the first time the regeneration, transfor - ing the application of gene editing techniques to carnation, mation, and genome editing of callus-derived shoots from two reports have been published until date. One described seed-propagated carnations that can consistently acquire the use of protoplasts but shoots were not obtained (Adedeji null segregants. Our finding is expected to contribute to the et al. 2024). In the other report, authors applied an electro- rapid breeding of new carnation varieties in the future. poration based approach to successfully produce mutations in the anthocyanidin synthase gene, but chimerism of the regenerated plants remained a challenge (Mori et al. 2024). Materials and methods Carnations are typically vegetatively propagated through cutting because most varieties are heterozygotes (Yagi Plant materials 2015). When heterozygotes are propagated by seed, traits segregate in the next generation and various character- Seeds of ‘Chabaud Giant’ were purchased from Fukukaen istics cannot be maintained. This is a disadvantage in the Nursery & Bulb Co., Ltd. (Aichi, Japan) and stored in a process of obtaining null segregants without foreign genes refrigerator at 4 °C until use (Fig S1). This variety blooms after genome editing. To prevent this, genome editing is with double, fringed, and medium-sized flowers, making it performed by transiently expressing foreign genes without suitable for early harvesting during outdoor cultivation. introducing them into the genome (Hamada et al. 2017; Adedeji et al. 2024) or by using homozygous lines for seed In vitro seed germination propagation. In genome editing using homozygotes, foreign genes can be removed by selfing to obtain null segregants Seeds were first surface-sterilized with 70% ethanol for with the original traits maintained. Since this material has 5 min, followed by 2% sodium hypochlorite with one drop not yet been tested in carnations, this study developed a of 10% Tween 20 for 15 min. They were then rinsed thrice genome editing system using seed-propagated variety. with sterilized distilled water. Twenty milliliters of solid To applied genome editing techniques, it is essential to medium containing Murashige and Skoog (MS) (1962) previously develop tissue culture procedures such as adven- basal salts, 30 g/l sucrose, and 8 g/l agar (FUJIFILM Wako titious shoot regeneration and transformation (Bekalu et al. Pure Chemical Corporation, Japan) were prepared and 2023). Callus-mediated (indirect) regeneration is advisable adjusted to pH 5.8 ± 0.1 before sterilization with an auto- since direct shoot regeneration often produces chimeras, clave at 121 °C for 20 min. Sterilized seeds were sown on 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 3 of 13 8 the medium in Φ90 × 19 mm Petri dishes. Seeds were cul- Rooting experiments tured in a growth chamber at 26 °C under a 16 h photoperiod provided by cool-white fluorescent lamps at a light intensity Newly induced shoots in 1.5 months after inoculation were of approximately 100 µmol/s/m . excised from explants or calli onto rooting medium at pH 5.8 supplemented with MS basal salts, 30 g/l sucrose, and Determination of the phytohormones involved in 4 g/l gellan gum, which facilitates observation of the roots callus induction better than agar. The medium included combinations of the following phytohormones: 0.01 mg/l KT, 5 mg/l GA ; Seeds sown in the medium germinated in about 3 days. 0.01 mg/l NAA, 0.01 mg/l IBA. To test the vessels, 50, 200, Subsequently, the top four true leaves were taken from each and 450 ml culture bottles were used, and the medium was seedling and cut into 5 mm square segments after 18 days filled to 40% of the vessel volume. Three shoots were placed of seedling culture. The segments were cultured in the same per bottle in triplicate. The culture environment was the medium used for germination but with phytohormones. same as that described in the former section “in vitro seed The experiment was divided into two parts. In experiment germination”. Rooting data was recorded after 1 month. 1, combinations of various hormones (IBA, NAA, 2,4-D, IAA, BA, TDZ, KT, zeatin, and 2iP) were used at 0.5 and Eec ff t of antibiotics on shoot regeneration 1.0 mg/l as callus induction medium (CIM). Each treatment combination was tested with 10 explants in triplicate. The The optimal antibiotic concentration for selection of trans- growth conditions were the same as those used for germina- formed tissues after Agrobacterium infection was deter- tion. After 1 month, the obtained calli were moved to a shoot mined. Kanamycin at 25, 50, 75, or 100 mg/l and hygromycin induction medium (SIM) supplemented with 0.1 mg/l NAA at 5.0, 7.5, 10.0, 12.5, or 15 mg/l were tested with a com- and 1.0 mg/l BA as described by Nontaswatsri et al. (2004). bination of 0.5 mg/l 2,4-D and 4.0 mg/l TDZ. All the other After 0.5 months, shoot induction rates were recorded, dis- components in the medium were the same as those used for tinguishing the CIM origin of the callus. 2,4-D as auxin seeds germination. Leaves of ‘Chabaud Giant’ were used and TDZ as cytokinin, which had high final shoot induc - as explants for culture under the same conditions as those tion rates, were selected as the hormones for CIM, and the described in the former section “in vitro seed germination”. detailed concentrations were reexamined as Experiment 2. Each treatment combination was tested with 10 explants in The combinations of 2,4-D and TDZ were examined at con- triplicate. The formation of callus and shoot were observed centrations of 0.01, 0.05, 0.1, 1.0, 2.0, 4.0, and 8.0 mg/l. The after 1 month. medium pH was adjusted to 5.8 ± 0.1 before sterilization at 121 °C for 20 min. The culture environment was the same as Cloning of DcPDS that described in the subsection above. The segments were cultured for 1.5 months. In addition, each treatment com- Genomic DNA of ‘Chabaud Giant’ was extracted from bination was tested with 10 explants in triplicate. Because leaves using the Nucleon PhytoPure Plant DNA Extract Kit this hormone combination yielded callus followed by shoot, (Cytiva, UK). Specific primers were designed based on the no independent SIM trials were conducted in Experiment 2. gene fragment in the carnation ‘Francesco’ database (Yagi et al. 2014). PDS genes were found in the Carnation DB Eec ff t of other factors in the induction of healthy and named DcPDS1, DcPDS2, and DcPDS3. Because the calli and shoots DcPDS2 and DcPDS3 sequences were identical, we later named them DcPDS2/3. We designed primers for the mid- Additional factors such as sucrose (20, 30, 40, and 50 g/l), dle and back regions of DcPDS1 and for the front and back agar (4, 8, 12, and 16 g/l), AgNO (0.5, 1.0, and 2.0 mg/l), regions of DcPDS2/3 (Table S1). The PCR reaction system and pH (5.0, 5.4, 5.8, and 6.2), were studied. The different was as follows: 10× PCR Buffer containing 5 µl KOD Plus treatments were compared with standard conditions based Neo, 5 µl dNTPs, 3 µl MgSO , 1 µl upstream primer, 1 µl on pH 5.8, as well as 8 g/l agar, 30 g/l sucrose, 0.05 mg/l downstream primer, 1 µl KOD plus Neo, 200 ng template 2,4-D, and 4 mg/l TDZ. Culture conditions were similar to DNA, and ddH O to complete the final volume. The reac - those described for seed germination. Each treatment com- tion conditions were as follows: 94 °C for 2 min; 98 °C for bination was tested with 10 explants in triplicate. 10 s, (Tm) °C for 30 s, and 68 °C for 30 s/kb, 45 cycles; and then hold at 25 °C. The PCR products were detected through 1.2% agarose gel electrophoresis. PCR product purification was performed using the GEL/PCR Purification Mini Kit (FAVORGEN) according to the manufacturer’s 1 3 8 Page 4 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 instructions. Next, purified PCR products were cloned with Biosystems), SeqStudio, or SeqStudio Flex 8 (Thermo the pTA2 vector, transformed into Escherichia coli DH5α, Fisher Scientific) automated DNA sequencing system using and identified via PCR. The positive clone was then selected the Prism Ready Reaction DyeDeoxy Terminator Cycle for sequencing. Sequencing kit 3.1 (Applied Biosystems Division, Perkin- Elmer, Foster City, CA, USA). Editing of genome sequence Construction of the CRISPR/Cas9 vector was confirmed using the CLC Main Workbench Version 20 sequence analysis programs (QIAGEN Aarhus A/S, Aarhus, Comparison of the PDS amino acid sequence of the carna- Denmark). tion with that of other plants revealed the first conserved domains in the second and fourth exons of DcPDS1 and DcPDS2/3, respectively (Fig S2). To knock out all three Results genes, 20 nt gRNAs were designed adjacent to the PAM sequence (Fig S3). Primers containing the AarI restric- Callus and shoot induction with different hormone tion site were annealed and ligated into pKIR1.1 (Tsutsui combinations and Higashiyama 2017). pKIR1.1 was a gift from Tetsuya Higashiyama (Addgene plasmid # 85758; h t t p : / / n 2 t . n e t / a d In the first experiment, four types of auxins were used in d g e n e : 8 5 7 5 8 ; RRID: Addgene_85758). A vector was con- combination with five cytokinins to select the appropriate structed and verified through standard cloning and sequenc - phytohormone combination for the induction of calli and ing, respectively. shoots from explants (Table 1; Fig S4). KT and IBA did not induce callus formation at most concentrations. Similarly, Transformation by Agrobacterium infection combinations of IAA with BA, KT, or 2IP did not induce callus hardly at all. The other 16 combinations induced calli Agrobacterium LBA4404 was used for the transformation efficiently, with induction rates ranging from 50.0 to 100%. experiments. Agrobacterium harboring the vector construc- However, many calli were either too small or hyperhydric tion for CRISPR/Cas9 was cultured in LB liquid medium (Fig S5A; S5B). Some of them induced roots during one supplemented with 100 mg/l rifampicin and 100 mg/l spec- month of culture in CIM (Fig S4), but none of them induced tinomycin at 28 °C for 2 d. A large-scale culture was per- shoots. Therefore, the callus was transplanted to the previ- formed until OD ≥ 0.6. The centrifuged pellet was then ously reported SIM supplemented with 0.1 mg/l NAA and adjusted to OD = 0.6–0.7 using fresh LB liquid medium 1.0 mg/ BA (Nontaswatsri et al. 2004). However, it was dif- supplemented with 0.1 µM acetosyringone and mercapto- ficult to obtain shoots from calli cultured in this SIM. In ethanol. The leaf segments were soaked in Agrobacterium 0.5 month after transplanting, shoots only grew from the suspension for 7 min, dried with filter paper, and placed onto two calli cultured in a medium supplemented with 0.5 mg/l MS solid medium supplemented with 0.05 mg/l 2,4-D and 2,4-D and 1.0 mg/l TDZ (Fig S5C). 4.0 mg/l TDZ. All the other components in the medium, and culture conditions were the same as those used for seeds ger- Callus induction with 2,4-D and TDZ mination. They were then co-cultivated for 3 d and moved to the selection medium containing antibiotics; 12.5 mg/l In the experiment 1 in the previous chapter, shoots were hygromycin and 50 mg/l meropenem. Each treatment com- induced only on media containing 2,4-D and TDZ. There- bination was tested with 10 explants in 80 replicates. The fore, as experiment 2, combinations of these phytohormones medium was changed every 2 weeks. Regenerated albino were tested at various concentrations to improve shoot for- shoots were selected as candidate genome-edited plants for mation efficiency. Callus formation was observed at all con - further analysis. centrations but decreased at 2,4-D concentrations > 1.0 mg/l (Fig. 1A). At a 2,4-D concentration > 2.0 mg/l, most calli Confirmation of the edited genome sequence were small, pale yellow, and became hyperhydric with- out shoot formation (Fig. 1A). The best conditions for the To determine the genome editing efficiency of DcPDSs, the induction of healthy green calli with shoots were 0.05 mg/l target sequence was amplified from gDNA using PCR and 2,4-D and 4.0 mg/l TDZ (Fig. 1B, Table S2). cloned into the standard sequencing vector (pSKII ) using an In-Fusion HD Cloning Kit (Takara Bio). The positive Eec ff t of other factors in callus and shoot induction clones were selected for Sanger sequencing. The nucleotide sequences were determined using the dideoxy chain-termi- To improve the shoot induction medium from the previ- nation method on 3130, 3130xl Genetic Analyzer (Appllied ous conditions (30 g/l sucrose, 8 g/l agar, pH = 5.8, absence 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 5 of 13 8 Table 1 Callus induction rates (%) from carnation leaf explants under differ ent phytohormone combinations Auxin (1.0 mg/l) Mean IBA NAA 2,4-D IAA Cytokinin BA 72.2 ± 20.0a 72.2 ± 20.0a 83.3 ± 9.6a 0.0 ± 0.0c 56.9 ± 11.9mn (0.5 mg/l) TDZ 72.2 ± 14.8a 77.8 ± 22.2a 72.2 ± 5.6a 83.3 ± 9.6a 76.4 ± 6.3mn KT 0.0 ± 0.0c 50.0 ± 19.2abc 88.9 ± 11.1a 0.0 ± 0.0c 34.7 ± 12.2n ZT 66.7 ± 9.6ab 100.0 ± 0.0a 88.9 ± 11.1a 77.8 ± 11.1a 83.3 ± 5.4 m 2IP 94.4 ± 5.5a 77.8 ± 11.1a 100.0 ± 0.0a 5.6 ± 5.6bc 69.4 ± 11.8mn Mean 61.1 ± 9.7xy 75.6 ± 7.6x 86.7 ± 4.0x 33.3 ± 10.7y Auxin (0.5 mg/l) Mean IBA NAA 2,4-D IAA Cytokinin BA 88.9 ± 5.6a 72.2 ± 20.0ab 100.0 ± 0.0a 0.0 ± 0.0d 65.2 ± 12.6mn (0.5 mg/l) TDZ 94.4 ± 5.6a 94.4 ± 5.6a 88.9 ± 5.6a 88.9 ± 5.6a 91.7 ± 2.5 m KT 5.6 ± 5.6d 61.1 ± 14.7abc 100.0 ± 0.0a 11.1 ± 5.6d 44.4 ± 12.2n ZT 88.9 ± 5.6a 94.4 ± 5.6a 94.4 ± 5.6a 27.8 ± 5.6 cd 76.4 ± 8.9mn 2IP 94.4 ± 5.6a 77.8 ± 14.7a 94.4 ± 5.6a 33.3 ± 9.6bcd 75.0 ± 8.6mn Mean 74.4 ± 9.5x 80.0 ± 6.1x 95.6 ± 2.0x 32.2 ± 8.5y Auxin (0.5 mg/l) Mean IBA NAA 2,4-D IAA Cytokinin BA 55.6 ± 5.6ab 88.9 ± 9.6a 88.9 ± 11.1a 0.0 ± 0.0c 58.3 ± 11.3 nm (1.0 mg/l) TDZ 94.4 ± 11.1a 100.0 ± 0.0a 83.3 ± 9.6a 77.8 ± 5.6a 88.9 ± 3.8 m KT 11.1 ± 11.1bc 72.2 ± 14.7a 83.3 ± 9.6a 0.0 ± 0.0c 41.7 ± 11.9n ZT 94.4 ± 5.6a 83.3 ± 9.2a 83.3 ± 9.6a 66.7 ± 1.7a 81.9 ± 5.6 m 2IP 94.4 ± 5.6a 94.4 ± 5.6a 88.9 ± 5.6a 5.6 ± 5.6c 70.8 ± 11.6mn Mean 70.0 ± 9.2x 87.8 ± 4.1x 85.6 ± 3.6x 30.0 ± 9.8y Data are expressed as the mean rates (%) of callus induction rates ± SE (n = 6, r = 3) after 1 month. Data followed by die ff rent letters are signifi - cantly die ff rent ( P < 0.05), as determined using Tukey–Kramer’s honestly signic fi ant die ff rence (HSD) test of AgNO ), various concentrations of sucrose, agar and Therefore, it was not necessary to include AgNO in the 3 3 AgNO , and pH values were investigated. The shoot induc- medium at the concentrations tested. tion rates of the base conditions were compared in the 2,4-D and TDZ concentration tests and in the tests of the other Root induction by different hormones and vessels components. Callus and shoot induction rates did not sig- nificantly differ when cultured at different concentrations Different hormones and vessels were evaluated to increase of sucrose or agar and under varying pH values, however, the rooting rate and reduce hyperhydricity. In 200 ml ves- hyperhydricity was variable (Table 2). In the sucrose treat- sels with GA fixed at 5 mg/l, different combinations of ment, rate of hyperhydric calli was significantly higher at a three phytohormones (NAA, KT, and IBA) at various con- concentration of 20 g/l. Based on the photos, 50 g/l sucrose centrations did not significantly affect in rooting rate from resulted in yellowish, brown, and fragile calli (Fig S6A). shoots and shoot hyperhydricity rate (Table 3). However, Agar tended to increase hyperhydricity at lower concentra- comparison of different volume vessels with fixing 5.0 mg/l tions, with 4 g/l being significantly lower than with 12 and GA and 0.01 mg/l IBA revealed that rates of rooted shoot 16 g/l (Table 2). Calli were dehydrated and their surface was the lowest significantly in 50 ml bottles, less than half appeared white at > 12 g/l agar (Fig S6B). Hyperhydricity of the 200 ml and 450 ml. In addition, the rate of hyperhy- tended to increase with advancing acidity and alkalinity, dric plantlets decreased with an increase in vessel volume. such as 5.0 and 6.2, and was significantly lower at 5.4 and The rate of hyperhydric plantlets was 22% and 90% in 450 5.8 than at 5.0. At pH 5.0, there were many light green calli and 50 ml bottles, respectively (Table 3). Based on these (Fig S6C). These results suggest that the base conditions of results, phytohormones were not critical, and larger vessel 30 g/l sucrose, 8 g/l agar, pH = 5.8 are adequate to reduce volumes, such as 450 ml, were more effective in maintain - hyperhydricity. As the concentration of AgNO increased, ing healthy shoots and promoting rooting. Some rooted the callus induction rate decreased (Table 2). In the AgNO - regenerants were successfully acclimated and potted in soil supplemented medium, callus hyperhydricity was sup- (Fig. 2). Through the above experiments, the following con- pressed, but shoots were not induced (Table 2; Fig.S6D). ditions were determined to obtain regenerants from seed- propagated carnations. For both callus and shoot induction, 1 3 8 Page 6 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Fig. 1 Effects of different concentration combinations of 2,4-D and explants inducing shoots at various concentrations of 2,4-D and TDZ TDZ on callus/shoot formation from carnation leaf segments. Photo- (B). Ten leaf segments were incubated as explants in each Petri dish graphs of calli/shoots in medium supplemented with 2,4-D and TDZ at (n = 10, r = 3). Photographs were taken 1 month after inoculation different concentrations ( A). Significant differences in the number of 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 7 of 13 8 Table 2 Callus / shoot induction rates under different medium compo - sitions other than phytohormones Treatment Callus induction Shoot induc- Rates of rates (%) tion rates (%) hyperhydric calli (%) Sucrose 20 86.7 ± 13.3 a 0.0 ± 0.0 a 53.3 ± 3.3 a (g/l) 30 96.7 ± 3.3 a 7.7 ± 3.3 a 13.3 ± 3.3 b 40 96.7 ± 3.3 a 3.3 ± 3.3 a 13.3 ± 3.3 b 50 96.7 ± 3.3 a 6.7 ± 6.7 a 16.7 ± 3.3 b Agar (g/l) 4 90.0 ± 5.8 a 10.0 ± 6.8 a 56.7 ± 23.3 a 8 96.7 ± 3.3 a 7.7 ± 3.3 a 20.0 ± 11.2 ab 12 96.7 ± 3.3 a 0.0 ± 0.0 a 0.0 ± 0.0 b 16 93.3 ± 3.3 a 7.7 ± 3.3 a 0.0 ± 0.0 b pH 5.0 90.0 ± 10.0 a 0.0 ± 0.0 a 43.3 ± 6.7 a 5.4 73.3 ± 17.6 a 0.0 ± 0.0 a 23.3 ± 3.3 b 5.8 93.3 ± 7.7 a 3.3 ± 3.3 a 16.7 ± 3.3 b 6.2 83.3 ± 12.0 a 0.0 ± 0.0 a 26.7 ± 3.3 ab AgNO 0.5 93.3 ± 6.7 a 0.0 ± 0.0 a 0.0 ± 0.0 a (mg/l) 1.0 76.7 ± 12.0 ab 0.0 ± 0.0 a 0.0 ± 0.0 a 2.0 40.0 ± 11.6 b 0.0 ± 0.0 a 0.0 ± 0.0 a Data are expressed as the mean rates (%) of callus / shoot induc- tion ± SE (n = 10, r = 3) after 1.5 months. Data followed by die ff r - ent letters are signic fi antly die ff rent ( P < 0.05), as determined using Tukey–Kramer’s HSD test leaf segments are placed on MS medium containing 30 g/l sucrose and 8 g/l agar at pH 5.8 as the basal medium, sup- Fig. 2 Appearance of regenerated carnation plantlets. Regenerants were obtained by inducing calli and shoots from leaf segments and plemented with 0.05 mg/l 2,4-D and 4.0 mg/l TDZ in petri rooting using established protocols. Healthy shoots (A) but not hyper- dishes. For rooting, shoots are grown on the basal medium hydric shoots (B) were acclimatized and transplanted into pots (C) containing the appropriate phytohormones (e.g., 5.0 mg/l GA and 0.01 mg/l IBA) in 450 ml culture bottles. screening. The antibiotic kanamycin was initially used to Genome editing using Agrobacterium-mediated select transformed shoots; however, many of the obtained transformation shoots were only non-transformants in white color. There- fore, hygromycin was selected based on the results of a pre- As we applied Agrobacterium-mediated transformation to vious study (Nontaswatsri et al. 2004). the regeneration system described in the previous section, When designing gRNA for the target DcPDS gene, it is we examined the antibiotic conditions for selecting trans- important to avoid the mutation sequence based on varietal formants. Calli were induced with < 50 mg/l kanamycin differences between the ‘Chabaud Giant’ material vari - or 10.0 mg/l hygromycin after 1 month of leaf segment ety and the ‘Francesco’ variety used for reference genome culture (Fig. S7A; S7B). Accordingly, 75 mg/l kanamycin sequences. In this study, we use PCR cloning to obtain two and 12.5 mg/l hygromycin were used for post-infection genome sequences for the DcPDS of ‘Chabaud Giant’. The Table 3 Rates of carnation shoots forming roots under different culture cond itions Phytohormone (mg/l) Vessel volume Rates of rooted shoots (%) Rates of hyperhydric shoots (%) (ml) GA NAA KT IBA 5.0 0.01 0.01 200 77.8 ± 11.1a 55.6 ± 11.1ab 5.0 0.5 0.5 200 66.7 ± 0.0ab 66.7 ± 19.3ab 5.0 0.5 200 66.7 ± 0.0ab 77.8 ± 11.1ab 5.0 0.01 50 33.3 ± 0.0b 89.9 ± 11.1a 5.0 0.01 200 77.8 ± 11.1a 55.6 ± 11.1ab 5.0 0.01 450 77.8 ± 11.1a 22.2 ± 11.1b Mean 66.7 ± 4.7 59.2 ± 6.4 Data are expressed as the mean rates of carnation shoots forming roots ± SE (n = 3, r = 3) after 1 month. Data followed by die ff rent letters are signic fi antly die ff rent ( P < 0.05), as determined using Tukey–Kramer’s HSD test 1 3 8 Page 8 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 resulting DNA sequences encoded both partial DcPDS1 ‘Chabaud Giant’ in this study. Calli were induced by most and full-length DcPDS2 or DcPDS3 (hereafter referred hormone combinations. However, particularly low levels to as DcPDS2/3). The lengths of the cloned DcPDS1 and of callus induction were observed when the auxin IAA was DcPDS2/3 were 2471 bp and 4337 bp, respectively (Fig. used (Table 1). IAA is a naturally produced plant hormone S8). The conserved regions of DcPDS1 and DcPDS2/3 that is rapidly degraded by many tissues and often cannot were designed for gRNA (Fig. S2). Albino shoots were pro- support the growth of cultured plant tissues (Hangarter et al. duced from the explants after infection with Agrobacterium 1980). In addition, the light provided by cool-white, fluores - containing the pKI1.1R vector (Fig. 3), although the shoot cent bulbs can promote the degradation of IAA. Therefore, production rate was only 2.5%. Sequence analyses of the IBA, NAA, and 2,4-D are more commonly used than IAA in albino shoots PCR amplicons revealed two different indels; plant tissue culture (Nissen and Sutter 1990; Stasinopoulos the deletion of one base (A) or the insertion of two bases and Hangarter 1990). Although the growth rate of calli was (AT) before the PAM site. Therefore, both indels provoked high under many hormone combinations, shoots were only a shift in the reading frame and a non-functional protein obtained from calli grown in a medium containing 2,4-D (Fig. 4). The probability of genome-edited shoots per regen- and TDZ. Furthermore, many calli were especially small erated shoots was 6.7%. Since the sequencing was limited and considerably hyperhydric, which may be the main rea- to albino shoots, it is possible that the normal shoots with son shoots were not obtained. Although 2,4-D is effective non-silenced PDS gene by in-frame editing were missed. If in callus induction (Maheshwari et al. 2011; Motte et al. that possibility is included, the true efficiency may be some - 2011), it is not typically effective in shoot induction (Han - what higher. garter et al. 1980). However, 2,4-D induced shoot regenera- tion in the present study, possibly because of the specificity among species or varieties. In shoot induction from carna- Discussion tion leaves, NAA is the most commonly used auxin (Frey and Janick 1991; Nakano et al. 1994; Abu-Qaoud, 2013), Tissue culture is an important step in the transgene, neces- with some use of IBA and IAA (Nontaswatsri et al. 2002; sitating the establishment of an induction protocol of callus Kanwar and Kumar, 2009). In carnations, TDZ has been and shoots (De Filippis 2014). Thus, combinations of four successfully used as a cytokinin for shoot induction from auxins and five cytokinins were evaluated to establish an leaves. With different varieties from ‘Chabaud Giant’, 0.1- in vitro regeneration protocol for homozygous carnations 4.0 mg/l of TDZ marked high shoot induction rates ranging Fig. 3 Appearance of plantlets or calli of carnation after genome plantlets without genome editing (A). Plantlets regenerated following editing. Regenerants were obtained from Agrobacterium-infected successful genome editing using the pKI1.1R vector containing the leaf fragments using established protocols. The carnation phytoene hygromycin resistance gene (B) desaturase gene (DcPDS) was targeted. Photograph show regenerated 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 9 of 13 8 Fig. 4 Altered nucleotide sequence of DcPDS by CRISPR/Cas9- than multiples of three cause frameshifts and the appearance of an derived genome editing. Electropherograms were obtained by sequenc- early termination codon or different amino acid. The sequences for ing PCR-cloned genome fragments for the wild-type (WT) and two gRNA and PAM are highlighted in yellow and light blue, respectively genome-edited strains (DcPDS1-KO and DcPDS23-KO). Indels other from 53 to 93% (Frey and Janick 1991; Nontaswatsri et al. the present study. Agar above 12/g drastically reduced the 2002; Abu-Qaoud, 2013). The concentrations used in this hyperhydricity rate (Table 2). However, the surface of the study were also in the range, suggesting that carnations are callus turned white and appeared to be low in water content, generally responsive to TDZ. inhibiting growth (Fig. S4). Previous studies have reported Different concentrations of sucrose and agar, as well that high concentrations of agar were less likely to induce as different pH values, were tested to obtain high-quality hyperhydricity, but were detrimental to shoot regeneration calli, reduce hyperhydricity, and improve shoot induction and inhibited growth, which supported our results (Arnold efficiency. Although the results showed no significant dif - and Eriksson 1984; Kadota et al. 2001; Suthar et al. 2011). ferences in shoot induction efficiency, they provided a basis The calli appeared more hyperhydric at pH 5.0 (Table 2; for selecting a more suitable medium. Shoot regeneration Fig. S6C). pH can affect the hardness of the agar medium: a occurred when the concentrations of supplemented sucrose lower pH softens the medium, whereas a higher pH hardens were more than 30 g/l (Table 2). Calli showed obvious hyper- it (Huang et al. 1995). Although shoots only grew at pH 5.8 hydricity in the medium supplemented with 20 g/l sucrose, in other media, there was no significant effect on the induc - and browning was observed when the sucrose concentra- tion rate or appearance of calli and shoots. Hyperhydricity tion was > 40 g/l. This result is similar to that observed in was thought to be due to a reduction in the amount of cel- previous research showing that increasing concentrations lulose and lignin required for cell wall lignification caused of sucrose can reduce hyperhydria, and that high sucrose by ethylene (Keverse et al. 1984). Silver ions can bind to concentrations can promote callus browning (Khosroushahi ethylene receptors and prevent the transduction pathway et al. 2011; Liu et al. 2017; Liang et al. 2019). Therefore, a (Gao et al. 2017). Therefore, AgNO has been used as an sucrose concentration of 30 g/l was the most appropriate in anti-hyperhydric agent in several plant species (Mayor et 1 3 8 Page 10 of 13 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 al. 2003; Vinoth and Ravindhran 2015). This study actually and sugarcane (Rastogi et al. 2018). Therefore, kanamycin showed that AgNO -supplemented medium suppressed cal- was unfavorable for the screening of PDS knockouts in lus hyperhydricity, but did not induce shoots unfortunately carnations. (Table 2; Fig. S6D). The carnation ‘Chabaud Giant’ used in Two studies on genome editing in carnations have this study may be too sensitive to AgNO , as even 0.5 mg/l recently been issued. One is a transient system using the AgNO was toxic too shoot differentiation. method of direct delivery of preassembled CRISPR/Cas9 Various hormones and vessels were evaluated to deter- ribonucleoproteins (RNPs) using protoplasts (Adedeji et al. mine the optimal rooting conditions. There was no signifi - 2024). Their method is superior in two aspects: elimination cant difference in rooting in the media containing different of chimerism and foreign gene-free, but organogenesis from phytohormones (Table 3). A few examples of rooting from protoplasts is difficult and shoots were not obtained. Another carnation shoots have been reported to be effective with study used a method of introducing RNPs into stem sections 1.0 mg/l IBA (Lu et al. 2021; Zheng et al. 2020) and 1.0 mg/l by electroporation (Mori et al. 2024). With this method, also NAA (Karami and Kordestani 2007; Ali et al. 2008; Zheng foreign DNA-free, they obtained regenerated shoots, but the et al. 2020). Although we did not test the effects of these problem of chimerism remained. We successfully devel- hormones alone in this experiment, it is likely that a num- oped a genome-editing system with a seed propagation-type ber of different auxins were effective to promote rooting carnation in which PDS was knocked out. Genome editing in carnations. The use of a large size vessels showed the of seed-propagated varieties established in this study will positive effect on root growth and reduced hyperhydricity accelerate carnation breeding as it will overcome the prob- (Table 3). It has been reported that nutrient supply increases lems of chimerism, persistence of foreign genes and trait and air ventilation was improved, all of which were condu- segregation, which are problems in vegetatively propagated cive to root growth and reduced hyperhydicity (Lai et al. varieties. 2005; Lynch et al. 2012). Some studies have shown that both ethylene and CO are accumulated in a closed vessel (reviewed by Polivanova and Bedarev 2022). In addition, Conclusions the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene (ACO) involving ethylene synthesis, has been In this study, a system for shoot regeneration from leaf shown to be significantly increased in hyperhydric plants. In explants of the homozygous carnation ‘Chabaud Giant’ was a well-ventilated environment, the accumulation of ethylene established. Of all the treatments tested, the best results and CO can be reduced to inhibit the hyperhydricity. of regeneration rate were reached 27.8% in MS medium Shoot induction efficiency was reduced following Agro- supplemented with 0.05 mg/l 2,4-D, 4.0 mg/l TDZ, 30 g/l bacterium infection, possibly because of altered levels of sucrose and 8 g/l agar and adjusted to pH 5.8 after 1.5 auxin and other hormones (Bettini et al. 2010; Mashigu- month. Moreover, the use of a large size vessels with 450 ml chi et al. 2019). The selection of knockout shoots for the volume was the best condition for root growth and reducing PDS gene by the antibiotic kanamycin was not success- hyperhydricity. Additionally, an Agrobacterium-mediated ful. This could be because it was difficult to recognize the transformation procedure was developed to obtain trans- difference in white color between the drug effect of kana - formed plants using hygromycin as the selection antibiotic. mycin and the loss-of-function effect of PDS. Practically Gene editing (CRISPR/Cas9) combined with the transfor- many white-colored regenerants were obtained as escapes mation/regeneration system was investigated using PDS as on the medium containing 75 mg/l kanamycin (Fig. S7C). the target gene. Sequencing analyses of the edited albino This indicates that carnations may be somewhat resistant to shoot revealed two different indels: one base deletion or kanamycin and shoots on the selection medium grow to a two bases insertion before the PAM sequence. These results certain stage. The calli on the medium containing kanamy- demonstrate that the carnation genome editing system was cin were transparent yellow (Fig. S7D), whereas those with established for the first time with a seed-propagation vari - hygromycin were healthy (green) (Fig. S7E). The results ety, which will provide convenience for the future study of show that even in the presence of antibiotic concentrations carnation gene function and the efficient breeding of new that weaken the explant, false-positive callus differentia - varieties. tion occurs with kanamycin, whereas it is suppressed with Supplementary Information The online version contains hygromycin. Albinism in non-transformed plantlets due to supplementary material available at h t t p s : / /d o i. o r g / 1 0 . 1 0 0 7 / s 1 1 2 4 0 - 0 kanamycin has been also reported in many plant species, 2 4 - 0 2 9 2 9 - 9 . such as Brassica juncea (Gao et al. 2020), Vigna mungo (Saini et al. 2003), Indian cowpea (Chaudhury et al. 2007) Acknowledgements The authors would like to thank Dr. Hiroshi Fukayama and Dr. Keiji Nishida for their advice on genome editing experiments. The authors thank Kaya Okamoto, Haruka Konishi, 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) (2025) 160:8 Page 11 of 13 8 Satoshi Kawamoto and Nanako Fukushima for their technical assis- genome editing. Int J Mol Sci 24(15):11920. h t t p s : / /d o i. o r g / 1 0 . 3 tance. The authors also thank Dr. Takako Narumi, Dr. Michio Kanechi, 3 9 0 / i j m s 2 4 1 5 1 1 9 2 0 and Dr. Hiroyasu Yamaguchi for their valuable discussions. Bettini P, Baraldi R, Rapparini F et al (2010) The insertion of the Agro- bacteriumrhizogenes rolC gene in tomato (Solanum lycopersicum L.) affects plant architecture and endogenous auxin and abscisic Author contributions ZJL performed tissue culture, Agrobacterium- acid levels. Sci Hortic 123(3):323–328. h t t p s : / /d o i. o r g / 1 0 . 1 0 1 6 / j mediated transformation, cloning, and sequencing of DcPDS genes; . s c i e n t a . 2 0 0 9 . 0 9 . 0 1 3 generated knockout transformants via genome editing; and drafted the Chaudhury D, Madanpotra S, Jaiwal R et al (2007) Agrobacterium manuscript. YU designed the experiments, analyzed the data, and re- tumefaciens-mediated high frequency genetic transformation of vised the manuscript. MY and RK analyzed the data and revised the an Indian cowpea (Vigna unguiculata L. Walp.) Cultivar and trans- manuscript. All the authors have read and approved the final manu - mission of transgenes into progeny. Plant Sci 172(4):692–700 script. De Filippis LF (2014) Crop improvement through tissue culture. Improvement of crops in the era of climatic changes. Springer, Funding Open Access funding provided by Kobe University. New York. https://doi.or g/10.10 07/97 8-1-4614-8830-9_12 This project was supported by JST SPRING (Grant No. JPMJFS2126) Fisher M, Ziv M, Vainstein A (1993) An efficient method for adventi - and partially supported by JSPS KAKENHI (grant Nos. 17K07654 tious shoot regeneration from cultured carnation petals. Sci Hortic and 23K05214). 53(3):231–237. https://doi.or g/10.10 16/03 04-4238(93)90071-W Frey L, Janick J (1991) Organogenesis in carnation. 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Plant HortScience 55(2):170–173. h t t p s: / /d o i .o r g /1 0 . 2 1 2 7 3 / H O R T S C I Cell Tissue Organ Cult 102135–102143. h t t p s : / /d o i. o r g / 1 0 . 1 0 0 7 1 4 3 3 4 - 1 9 / s 1 1 2 4 0 - 0 1 0 - 9 7 1 3 - 9 Zheng L, Xiao Z, Song W (2020) Effects of substrate and exogenous Publisher’s note Springer Nature remains neutral with regard to juris- auxin on the adventitious rooting of Dianthus caryophyllus L. dictional claims in published maps and institutional affiliations. 1 3

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"Plant Cell, Tissue and Organ Culture (PCTOC)" – Springer Journals

Published: Jan 1, 2025

Keywords: CRISPR/Cas9; Stable transformation; Homozygote; Hyperhydricity; Callus induction; Shoot induction

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